Genotyping Protocols for Genetically Engineered Mice

Abstract

Historically, the laboratory mouse has been the mammalian species of choice for studying gene function and for modeling diseases in humans. This was mainly due to their availability from mouse fanciers. In addition, the short generation time, size, and minimal food consumption compared to that of larger mammals was a definite advantage. This led to the establishment of large hubs for the development of genetically modified mouse models such as the Jackson Laboratory. Initial research into inbred mouse strains in the early 1900s revolved around coat color genetics and cancer studies, but gene targeting in embryonic stem cells, the introduction of transgenes through pronuclear injection of a mouse zygote, along with current CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA gene editing have allowed for easy manipulation of the mouse genome. Originally, to distribute a mouse model to other facilities, standard methods had to be developed to ensure that each modified mouse trait could be consistently identified no matter which laboratory requested it. This task became easier to establish uniform protocols with the development of the polymerase chain reaction (PCR). This chapter will provide guidelines to identify genetically modified mouse models, mainly using endpoint PCR. In addition, we will discuss strategies to identify genetically modified mouse models that have been established using newer gene editing technology such as CRISPR.

Isolation and purification of genomic DNA from mouse tissues and early-stage embryos

Basic Protocol 1: Isolation and purification of genomic DNA using phenol and chloroform

Alternate Protocol 1: Digestion with proteinase K followed by crude isopropanol extraction for tail biopsy, and ear punch samples

Basic Protocol 2: Purification of DNA using a semi-automated device

Basic Protocol 3: Purification of DNA from semen, blood, or buccal swabs

Basic Protocol 4: Purification of DNA from early-stage mouse embryos to assess CRISPR gene editing

PCR-based strategies to identify Genetically Engineered Mice

Basic Protocol 5 Routine endpoint PCR-based genotyping using DNA polymerase and a thermal cycler

Basic Protocol 6: T7E1/Surveyor assays to detect indels (insertion or deletions) following CRISPR editing

Basic Protocol 7: Detection of off-target mutations

Basic Protocol 8: Deletion of genomic sequence with a pair of guide RNAs

Basic Protocol 9: Detecting gene knock-in events following CRISPR editing

Basic Protocol 10: Screening of conditional knockout floxed mice

Introduction

The development of Genetically Engineered Mice (GEMs) depended on the production of inbred mouse lines and the rediscovery of Mendel’s classic inheritance laws. Early geneticists realized that creation of inbred mouse lines where all mice would have identical genomes would be beneficial to study inheritance like pea plants studied by Mendel. As small mammals with a fast generation time and shared mammalian characteristics with humans, inbred mouse models became ideal to characterize mutations related to diseases in people. Most of the early mutation analysis in mice was related to observable changes in coat color (the earliest phenotyping) such as the Agouti locus (Silver, 1995). Some naturally occurring mutations such as those causing obesity in mice were identified (Austin et al., 2004), but early genetic research in mice often involved using ionizing radiation to create heritable mutations. Analyses to identify the genetic makeup or genotype of the various mutant alleles (e.g., homozygotes, heterozygotes) involved creating linkage maps to determine relative cross-over distances between genes. Much later, physical maps were developed by fragmenting DNA using restriction enzymes to create relatively smaller fragments that could be sequenced with techniques such as Sanger sequencing (developed in 1977). With the physical map of certain genes established, large centers for mouse genetics and modeling such as the Jackson Laboratory could distribute their mice to other facilities and have uniform methods to genotype these mutant mouse colonies.

Even though fruit flies (Drosophila melanogaster) have also allowed significant advances in genetic research (especially as they share 75% of the same human disease-causing genes), the use of mice in studying genetics has still been needed for understanding and modeling complex diseases. Overall, mouse models have been essential in immune, cancer, and neuronal research particularly with mice, as mammals, being biologically similar to humans. Human and mouse lineages diverged about 75 million years ago, yet, even with the mouse genome being about 14% smaller than the human genome, there is large-scale synteny between the two genomes (Mouse Genome Sequencing Consortium, 2002). While only 40% of the human genome can be aligned to the mouse genome at the sequence level, 99% of mouse genes share a human homologue. Only 300 genes are unique to either species (Mogil, 2019). Nonetheless, the mouse ENCODE project (ENCyclopedia of DNA Elements; http://mouseencode.org) has shown that there are differences in how these genes are regulated (Mouse ENCODE Consortium, et al., 2014). In particular, the regulatory elements of genes linked to the immune system, metabolic processes, and stress responses appear to differ most between mice and humans.

A major breakthrough in mouse genetic research came with the ability to make a specific mutation within a targeted genetic allele in mice, where gene editing greatly accelerated research via the generation of mouse models. Gene knockout mice, for example, were able to be generated through homologous recombination in embryonic stem cells (Hall et al., 2009, Limaye et al., 2009). The knockout mouse project (KOMP), which employs either conventional gene targeting or gene trapping techniques to disrupt genes, was eventually initiated to create a knockout for every mouse gene (Austin et al., 2004). Another important step in mouse research was through the microinjection of a foreign transgene into the mouse zygote, which essentially complemented the knockout by allowing for gene overexpression (Cho et al., 2009, Haruyama et al., 2009). However, CRISPR-mediated gene editing through microinjection of Cas9, guide RNAs, and, at times, a donor DNA was later able to supplant conventional gene knockout and knock-in techniques performed in ES cells (Hall et al., 2009). Initially, the identification of a targeted mutation, whether through a knockout mutation or an inserted transgene, was achieved through Southern blot (Brown, 2001). The genotyping of various mutant mouse colonies was later simplified with the development of the polymerase chain reaction (PCR) by Kary Mullis in 1987. PCR simplified the task of copying specific genetic sequences using oligonucleotide primers, heat stable DNA polymerases, and equipment to quickly heat and cool and extend DNA templates. This technology helped to distribute these genetically modified mice from a few large centers to virtually any other mouse facility (see internet resources).

Isolation and purification of genomic DNA from GEM:

Basic Protocol 1: Digestion with proteinase K then purification using phenol/chloroform

Alternate Protocol 1: Digestion with proteinase K followed by crude isopropanol extraction for tail biopsy, and ear punch samples

Basic Protocol 2: Genomic DNA purification using an automated system

Basic Protocol 3: Genomic DNA purification from semen, blood, or buccal swabs

Basic Protocol 4: Genomic DNA purification from mouse blastocysts following CRISPR editing

Basic Protocol 1: Digestion with proteinase K then purification using phenol/chloroform

Isolation of mouse genomic DNA using phenol/chloroform (see also DNA isolation protocol from Jackson Laboratories under internet resources and phenol-chloroform-isoamyl alcohol (PCI) DNA extraction (Barker et al.,1998).

Commercially available DNA extraction kits are available from different vendors that typically involve silica spin columns, yet traditional DNA extraction methods through either phenol/chloroform or precipitating DNA out of solution are still commonly used. Phenol/chloroform extraction can be labor-intensive and involves hazardous chemicals but may be preferred in terms of cost and high yield pure genomic DNA.

Materials

Mouse tail biopsies (<0.5 cm) depending upon Institute Animal Care and Use Committee (IACUC) guidelines

Mouse ear punch biopsies (from the pinna of the ear); Ring handle ear punch, 5mm (Roboz Surgical Instrument Co., cat. no. 65–9902)

Reagents and Solutions

Tris-HCl (Trizma hydrochloride solution, 1 molar (M), pH 7 (Millipore-Sigma, cat. no. T1819)

Ethylenediaminetetraacetic acid (EDTA), 0.5M, pH 8 (Millipore-Sigma, cat. no. 03690)

Sodium dodecyl sulfate (SDS) solution, 20% (Millipore-Sigma, cat. no. 05030)

Sodium Chloride (NaCl) solution, 5M, (Millipore-Sigma, cat. no. S5150)

Proteinase K powder, 100 milligrams (mg), make a 10 mg/ml solution in water and store at 4°C (Thermo Fisher Scientific, cat. no. AM2542)

Chloroform, 500 milliliters (ml), (Fisher Scientific, cat. no. MK-4440–500)

Isopentyl (Isoamyl) alcohol, 500 ml, (Fisher Scientific, cat. no. 18–611-112)

Ultrapure buffer-saturated phenol, 100 ml, (Thermo Fisher Scientific, cat. no. 15513–039)

Isopropyl alcohol, 500 ml, (Fisher Scientific, cat. no. 02–003-132)

TE Buffer, Tris-EDTA, 1X Solution, pH 7.4, (Fisher Scientific, cat. no. BP2476100)

Eppendorf thermomixer F1.5 (Eppendorf, cat. no. EP5384000020) or equivalent

Microcentrifuge (e.g., Eppendorf cat. no. 5425) or equivalent (capable of at least 15,000 x g)

DeNovix Spectrophotometer/Fluorometer, (DeNovix, cat. no. DS-11) or equivalent

SpeedVac DNA130 Vacuum Concentrator, (Thermo Fisher, cat. no. DNA130–115), or equivalent

Lysis buffer stock solution:

Final concentration (in distilled water) can be stored up to 1 year at room temperature.

100 mM Tris-HCl

5 mM EDTA

0.2% SDS

200 mM NaCl

Add Proteinase K (10 mg/ml in water) such that the final concentration is 500 μg/mL

Phenol/chloroform/isoamyl alcohol solution (25:24:1):

Combine chloroform with isoamyl alcohol, at a 24 ml chloroform: 1 ml isoamyl alcohol concentration in a 50 ml polypropylene tube. Then add 25 ml of phenol for a final volume of 50 ml.

Caution: use eye protection, gloves and other appropriate personal protective equipment (PPE) as this and subsequent steps contain volatile, and corrosive chemicals.

Wrap the polypropylene tube in aluminum foil for long term storage, up to 2 months, as light degrades the solution.

1. Place each tail biopsy in a microcentrifuge tube with 0.5 ml of the lysis buffer plus Proteinase K (see recipe).

2. Transfer the microcentrifuge tube into a thermomixer to digest the tail biopsy (set at 55°C and use mixing, at least 500 rpm).

3. Incubate the tube in the thermomixer for a minimum of 4 hours, or preferably overnight.

   After incubation, there should be a semi-clear fluid layer on top of a layer of undigested mouse hair. This indicates complete digestion of the tail biopsy sample.

4. Transfer the semi-clear liquid tail digestion (from step 3) to a new microcentrifuge tube.

5. Add 0.5 ml of the phenol/chloroform mix and vortex carefully to create an emulsion.

6. Centrifuge the tube in a table-top centrifuge at room temperature (at full speed, about 13,000 rpm, [15,871 x g]) for 10 minutes.

   The aqueous layer containing DNA and RNA, will be on top of the hydrophobic layer, which contains lipids and other unwanted cell debris.

7. Transfer the aqueous upper layer to a new microcentrifuge tube.

8. Add 0.5 ml of the chloroform/isopentyl alcohol to the tube and carefully vortex mix the tube.

9. Place the tube in a centrifuge and again spin at 13,000 rpm for 10 minutes.

10. Transfer the upper aqueous layer to a new microcentrifuge tube.

11. Add 0.7 ml 100% ethanol to the tube and vortex. A stringy white to semi-clear precipitate should be evident.

12. Centrifuge the tube in a table-top centrifuge at room temperature, at 13,000 rpm, for 10 minutes.

    A viscous semi-clear pellet should be observed at the bottom of the microcentrifuge tube

13. Carefully remove the ethanol so as to not dislodge the DNA pellet.

14. Wash the pellet with 70% ethanol in water (about 0.25 ml) to remove any trace phenol and chloroform.

15. Centrifuge the tube in a table-top centrifuge at maximum speed for about 2 to 5 minutes.

16. Carefully remove the ethanol.

17. Dry the pellet by using a speed-vacuum concentrator or allow the pellet to air dry. The pellet will appear to be white or a clear gel-like pellet after drying.

18. Dissolve the pellet in 1X TE, ~ 250 – 400 μl depending upon the size of the pellet.

19. Place the tube in the thermomixer at 55°C for about 1 hour to allow the purified DNA to dissolve in solution. No remanent of the pellet should be observed. Ensure that there is an even distribution of the purified DNA throughout the solution.

20. Check the concentration and quality of the DNA using a spectrophotometer (see Figure 1A and 1B).

    Depending upon the volume of the DNA solution, the final concentration is usually about 100 nanogram (ng) per μL. If possible, check the ratio of absorbance at 260 nm (nanometers) to 280 nm. A value of 1.9 is optimal. A ratio of 2.0 indicates the presence of RNA (which should have been degraded by the addition of isopentyl alcohol). The ratio of absorbance at 260 nm to 230 nm should be 1.9 indicating minimal organic solvent contamination.

21. Store the genomic DNA at 4°C rather than freezing to avoid ice crystals from shearing the genomic DNA.

Figure 1 (A, B):

A) Graphical display of output from the spectrophotometer. Absorbance units on the Y axis and wavelength in nanometers on the X axis. B) Abbreviated .csv file output displaying the absorbance reading at 260 nm and ratio of absorbance at 260 nm to 280 nm (nucleic acid: protein). The 260/230 ratio is an indication of nucleic acid to other organic contamination.

Alternate Protocol 1: Digestion with proteinase K followed by crude isopropanol extraction for tail biopsy, and ear punch samples

This protocol describes crude purification of DNA from mouse tail DNA based on Laird et al., (1991).

1. Tail biopsy digestion should be as described in steps 1 through 4 of Basic Protocol 1.

2. The semi-clear digest should be transferred to a new centrifuge tube.

3. An equal volume of 100% isopropanol should be added (approximately 0.5 ml).

   A semi-viscous transparent layer should be evident.

4. Vortex mix the solution and a stringy white DNA precipitate should appear.

5. Using a sterile pipette tip, spool the crude DNA onto the tip.

6. Allow the DNA to air dry by inverting the tip to allow the excess isopropanol to drain and evaporate

7. Add about 0.25 ml of 1X TE buffer to a new centrifuge tube.

8. Once the DNA on the pipette tip has dried, place the tip into the tube containing 1X TE buffer.

9. Allow the DNA to “rehydrate” in the 1XTE buffer.

10. Heat the tube in a thermomixer to 37°C for about 1 hour to fully dissolve the genomic DNA.

11. Check the concentration and quality of the DNA using a spectrophotometer (as in step 20 of Basic Protocol 1).

    Depending upon the volume of the DNA solution a good concentration is 100 nanogram (ng) per μL. If possible, check the ratio of absorbance at 260 nm to 280 nm. A value of 1.9 is optimal.

12. Store the genomic DNA at 4°C.

Basic Protocol 2: Genomic DNA purification using an automated system

This protocol describes purification of genomic DNA from mouse tail or ear tissue using an automated device.

Materials

Maxwell 16 instrument (Promega Corporation, Madison WI) see internet resources

Maxwell 16 Tissue DNA purification kit (cat. no. AS1030, Promega Corporation, Madison WI)

Mouse tail biopsies (<0.5 cm) depending upon Institute Animal Care and Use Committee (IACUC) guidelines

Mouse ear punch biopsies (from the pinna of the ear); Ring handle ear punch, 5mm (Roboz Surgical Instrument Co., cat. no. 65–9902)

Note: Promega will discontinue the Maxwell 16 instrument and purification kits by February 2024. The substitute instrument is the Maxwell RSC (cat. no. AS4500) and associated purification kits are (cat. no. AS1880).

This instrument uses mechanical pistons to physically disrupt tissue in a lysis buffer and paramagnetic particles to separate nucleic acid from other components of the dissociated tissue. This system has two components that work together to purify genomic DNA in parallel for up to 16 separate pieces of mouse tissue. The first component is the instrument itself. It contains a row of 16 metal pistons which fit into a plastic, disposable plunger and physically agitate and disrupt the tissue sample. The second component is a disposable cartridge of wells that contain a lysis buffer, buffer containing the magnetic particles, and an elution buffer to separate the magnetic particle/nucleic acid combination from the remaining dissolved components. The cartridges are lined up in a tray that are positioned under each piston. Purification from up to 16 separate tissue samples can be performed in a semi-automated “hands off” manner in approximately 45 minutes.

1. The default setting for purification of genomic DNA from tissue is SEV (Standard Elution Volume)

2. Fill the provided blue elution cartridges with 250 to 300 μL of the provided elution solution. Each blue cartridge will contain the final purified nucleic acid solution.

3. Remove the foil cover on each supplied Maxwell 16 cartridge.

   Each cartridge contains a series of 7 wells containing various solutions to aid in lysis of tissue samples and 1 well containing paramagnetic particles in suspension. One cartridge is used for each tissue sample in parallel for up to 16 biopsies. Each cartridge is placed in the vertical position with the plastic notch final well at the bottom (see Figure 2)

4. The disposable plastic plunger (supplied with the kit) is placed into the last well.

5. Switch on the Maxwell 16 device and Press Menu from the operational screen

6. Press “Run/Stop”

7. Using the up/down button scroll and select DNA from the options list and press “Run/stop.”

8. Select tissue from the next menu.

9. Select the Run/stop button and follow the instructions to open the door.

10. Place the corresponding blue cartridge(s) containing elution buffer into the silver, metal holding rack.

11. Place the corresponding purification cartridge(s) into the inner chamber so that they line up in the same position as the blue elution cartridges.

12. Select run/stop and the instrument will automatically move the deck into the inner chamber.

13. The menu will prompt the user to close the door.

Figure 2:

Maxwell 16 automated DNA purification device showing the device and the associated Purification cartridge (white) and elution tube (blue). The provided elution solution is placed into the blue cartridge. The plastic film is removed from each cartridge (in white). A plastic piston (provided in the kit) is placed in the well nearest to the elution tube. The mouse tissue biopsy sample is placed in the well furthest from the elution tube.

At this point the device is entirely “hands-off”. At each stage the screen will indicate the current step in the purification process. The metal prongs will pick up the plungers in a coordinated sequence based upon the default program. The program is a series of lysing, magnetic capture of particles, binding, washing, drying, and final elution into the blue elution cartridges. An audible message will indicate when the process is complete, and the purified DNA is deposited into the blue cartridge. A typical run takes 45 minutes to complete purification of between 1 to 16 tissue samples in parallel. The user can then transfer the eluate to a separate tube for downstream application such as quantitation in a spectrophotometer and PCR amplification. Occasionally, some of the dark paramagnetic particles can persist in the final eluate. This does not affect the quality of the template DNA for standard, endpoint PCR.

Basic Protocol 3: Genomic DNA purification from semen, blood, or buccal swabs

This protocol details purification of genomic DNA from mouse tissue, blood, semen, and buccal swabs using alkaline lysis buffer and heat (HotSHOT method: Truett et al., (2000), and Mitrecic et al.,2008)).

Materials

Mouse tail biopsies (0.2 cm or less) depending upon Institute Animal Care and Use Committee (IACUC) guidelines

Mouse ear punch biopsies (from the pinna of the ear); Ring handle ear punch, 5mm (Roboz Surgical Instrument Co., cat. no. 65–9902)

PCR Tube Strips, 0.2ml (Eppendorf, cat. no. 951010022) or equivalent

BRAND 96-well Plates (Fisher Scientific, cat. no. 14–380-803) or equivalent

1. The tissue biopsy, approximately 0.2 cm or less) is placed in 75 μL of alkaline lysis solution in a 0.2 ml thin-walled PCR tube or well of a 96-well plate.

2. The tubes or plates are heated in a thermal cycler to 95°C for 10 minutes to 1 hour.

3. The tubes or plate are then cooled to 4°C.

4. The alkaline solution is neutralized with an equal volume (75 μL) of neutralizing reagent.

5. Quantification of the concentration of DNA can be performed as in step 20 of Basic Protocol 1.

Depending upon the concentration, 5 μL of the crude lysate per 25 μL PCR reaction is sufficient for PCR-based amplification.

Basic Protocol 4: Genomic DNA purification from mouse blastocysts following CRISPR editing

Generally, the CRISPR gene editing result can only be confirmed from the DNA of live pups once they are born or weaned. As the animal’s gestation and weaning date could add up to several weeks, the whole process can take time. Therefore, to optimize the generation of CRISPR transgenic animals, testing the fidelity of genome editing by specific guide RNA(s) in mouse blastocysts can be beneficial. The following procedure will demonstrate a simple, and efficient method for genomic DNA extraction from a single blastocyst 4 days after CRISPR editing of the gene of interest.

Materials

Collection of blastocysts

Microcapillary (World Precision, cat. no. 1W-150)

Aspirator tube assembly (Sigma-Millipore, cat. no. A5177)

KSOM (Sigma-Millipore, cat. no. MR-106)

M2 (Sigma Millipore, cat. no. MR-106)

Blastocyst Lysis Buffer (See reagents and solution for the recipe)

1M Tris-HCl (pH8.0) (Teknova, cat. no. T1115) or equivalent

KCl (Sigma Millipore, cat. no. P3911) or equivalent

Gelatin (Sigma Milipore, cat. no. G2500) or equivalent

NP40 (Thermo Scientific, cat. no. 85124) or equivalent

Tween20 (Sigma Millipore, cat. no. 11332465001) or equivalent

Proteinase K (Thermo Scientific, cat. no. E00491)

Distilled water (Quality Biological, cat. no. 351–029-721) or equivalent

Lab equipment

Mastercycler, nexus gradient (Eppendorf, cat. no. 6331) or equivalent

Lab Supplies

PCR Tube Strips, 0.2ml (Eppendorf, cat. no. 951010022) or equivalent

60mm culture dish (Corning, cat. no. 430589) or equivalent

4 well culture plate (Corning, cat. no. Z688746) or equivalent

1. Blastocysts should be collected after 4 days of in vitro culture following the injection of guide RNA/Cas9 complex. During the 4 days, the embryos are cultured in KSOM media in a cell culture incubator (37°C, 5% CO₂).

2. On day 4, pre-incubate M2 media in a cell culture incubator (37°C, 5% CO₂) for 10–20 minutes. Then prepare a 500 μl M2 drop in a 60mm culture dish.

3. Transfer blastocysts into the M2 drop in a four well plate using the aspirator tube assembly.

4. Using the microcapillary assembly, each blastocyst is transferred to the bottom of PCR tube, care should be taken to minimize the amount of M2 media inside the tube.

   The blastocysts in the PCR tube can be stored at −20°C until extraction of genomic DNA

5. Add 10 μl of blastocyst lysis buffer into each PCR tube, place the PCR strip into the Mastercycler.

6. Set the program in the Mastercycler to 56°C for 30 minutes, then increase the temperature to 95°C for 10 minutes. Allow the PCR tubes to cool to room temperature.

7. Use 5 μl of the extraction as template for PCR reaction of 30 μl in total.

PCR-based methods for genotyping mice

Basic Protocol 5: Routine endpoint PCR-based genotyping using DNA polymerase and a thermal cycler.

Basic Protocol 6: T7E1/Surveyor assays to detect indels (insertion or deletions) following CRISPR editing

Basic Protocol 7: Detection of off-target mutations

Basic Protocol 8: Deletion of genomic sequence with a pair of guide RNAs

Basic Protocol 9: Detecting gene knock-in events following CRISPR editing

Basic Protocol 10: Screening of conditional knockout floxed mice

Introduction

The term genotype refers to the complete set of instructions for a particular gene. Genotyping is the process of identifying one particular gene, and how it differs from other genes. In contrast, the phenotype is the expression of the gene in the form of a protein or regulatory process. The focus of this module is to present methods of distinguishing a normal or “wild-type” gene from the mutant or modified gene in genetically modified mouse models (GEMs). One such method is to use endpoint Polymerase Chain Reaction (PCR) to increase the number of gene segment copies to a point where this signal can be detected (Mullis et al.,1987). This module will not be an exhaustive description of all the flavors of PCR. For example, quantitative PCR can be used to define differences in the expression of a gene using fluorescent indicator dyes such as SYBR green, or using digital PCR when the absolute copy number of a gene needs to be determined. This module will mainly highlight simple endpoint PCR methods needed to amplify and distinguish modified mouse genes from wild type gene sequence.

The first step in this process is to have a clear physical map of the gene sequence and its position in the context of neighboring genes. There are several online browsers to identify your gene of interest (GOI). One example is the National Center for Biotechnology Information (NCBI) gene browser (see internet resources). In the search field, one types in the model organism and gene name or established gene ID. The search will hyperlink to several known aliases and model organisms. The browser will then show the position relative to the appropriate chromosome and show the physical map with the known start site of the coding sequence, introns, exons, and stop sequences. This gene information is also useful if any alternate protein products or splice sites are known.

The process for amplifying gene segments (Basic Protocol 5) can be divided into three phases: a) denaturation of double stranded DNA, b) annealing primers, and c) extension of the nucleotide sequence. Each phase is repeated in cycles with logarithmic doubling of the sequence until the reagents that catalyze the reactions deplete. The main starting components to any PCR reaction are the DNA template, short nucleotide sequences complementary to the DNA template (primers), deoxy-nucleotide phosphate (a mix of four bases), and a heat stable DNA polymerase to extend each gene segment copy. The DNA template for PCR-based genotyping is generally purified genomic mouse DNA. Methods to isolate and purify mouse genomic DNA are described in Basic Protocols 1 through 4.

Other components of the basic PCR reaction

PCR buffers

Almost all basic PCR protocols require the addition of a buffer solution. This solution like many other enzymatic reaction solutions, contains a Tris-based solution and potassium chloride (KCl). These buffers moderate the pH when subjected to varying temperatures, which otherwise would be detrimental to the structure and function of the polymerase enzyme. Generally, most suppliers use Taq (Thermus aquaticus) polymerase and will supply a reaction buffer that is optimized for the DNA polymerase. The buffer is generally supplied as either a 5- or 10-times concentrated solution and is then diluted to a final concentration (1X). Most buffers contain magnesium chloride (MgCl₂) where the concentration has been optimized for the specific polymerase.

Magnesium solution (divalent cation)

Taq polymerase activity is dependent upon the presence of free divalent magnesium (Mg²⁺). Most current commercially available PCR buffers include MgCl₂ in the solution at a concentration that is optimized for that polymerase. However, one can also purchase magnesium-free PCR buffers and supplement this buffer with MgCl₂ if the desired amplicon is not observed. However, excess Mg²⁺ can also lead to increased non-specific amplicons.

DNA polymerase

Since the initial discovery and development of the first generation of DNA polymerases, there have been many engineered variations of Taq that can be used when the amplicon is long, for GC rich PCR products, or to reduce the generation of non-specific bands (e.g., Hot start Taq, or antibody conjugated Taq). Additionally, a high fidelity Taq with good proofreading activity is suggested for PCR when either subcloning the amplicon into a vector, or when sequencing will be performed to confirm the proper amplified product for genotyping purposes (this includes Pfu DNA polymerase from Promega, or KOD DNA polymerase from Sigma-Aldrich). For routine PCR, where the amplicon is less than about 1000 base pairs and sequence fidelity is not needed, Taq polymerase is generally sufficient. Most often Taq polymerase is supplied as a concentrated solution in a glycerol base to prevent shearing of the enzyme by ice crystals during cold storage. For example, ExTaq (from TakaraBio) is supplied as a 5 unit/μL stock solution and is diluted to a 1-to-1.25-unit concentration for a 50 μL final reaction volume. To reduce un-wanted amplicons, hot start Taq has also been developed. In this variation of Taq, the enzyme is modified such that it is only active at high temperatures (non-specific templates can be extended in the ramping process to achieve the denaturation phase of the PCR reaction).

Method 1: Identification of primer sequences using MacVector

Primers are short nucleotide sequences (generally 18–20 base pairs), which are complementary to the gene segment to be amplified. In a typical PCR reaction, there are generally two to four primers which flank the gene region to be amplified starting with binding to the “sense” and, in the opposite direction, the “anti-sense” strand of genomic DNA. There are quite a few software design packages that can be used to identify primers for PCR based on several search criteria such as the annealing (binding to the DNA template) temperature, self-complementarity, and factors such as % GC (Guanine/Cytosine) content, which can affect the annealing of primers to the DNA segment. In general primer length should be between 15 and 30 nucleotides for routine amplification. The GC content (affects the annealing of each primer to the template) should be between 40–60%. The 3’ end of the primer ideally should be either a G or C base to increase binding to the template DNA strand. Self-complementarity should be avoided as a “hairpin” shape can occur preventing proper binding to the template strand. The annealing temperature of each primer should approximate each other especially if a third primer is used to distinguish the zygosity of each mouse to be genotyped (see Figure 3).

Figure 3(A,B):

Graphical output from MacVector displaying optimal amplicons following the pick 5’ and 3’ entry fields. Figure 3B: Primer report from MacVector displaying both primer sequences, sequence position, and calculated annealing temperature.

Genetically engineered mutations in mice can vary from small CRISPR mediated indels to the insertion of full transgenes that have a promoter, protein coding cDNA, and polyA (Hall et al.,2018 and Haruyama et al.,2009). Genetic sequence from the NCBI (National Center for Biotechnology Information) website should be used to piece together the desired mutation when creating GEM. This is needed whether generating guide RNA to target a selected genetic locus, designing donor DNA for homology directed repair (HDR), or with the assembly of a transgene.

CRISPR gene editing

Indels are commonly identified with the T7E1 enzyme mismatch assay (Hall et al. 2018), which are generated with repair of a CRISPR mediated double strand break (DSB). The introduction of a point mutation or small protein tag like 6x His, on the other hand, involves HDR via a donor DNA following the creation of a DSB. In this case, the genotyping strategy typically is planned around the engineered introduction/ablation of a restriction enzyme site leading to restriction fragment length polymorphism (RFLPs) analysis of the PCR product. Both PCR methods use flanking primers that are designed around the desired mutation. For large genetic insertions, however, one of the primers may need to be created to target the inserted sequence while the opposite primer will bind to sequence flanking the insertion.

Transgene

Transgenic mouse creation often encompasses the introduction of a foreign sequence (such as Cre [cyclization recombinase] from a bacteriophage, green fluorescent protein [GFP] from jellyfish, a human cytomegalovirus [CMV] promoter, or even a bovine growth hormone polyA) into a mouse (Haruyama et al., 2009). To design a PCR strategy to genotype transgenic mice, the sequence of the transgene should be known so that it can be entered into any primer design software package. The microinjected transgene is generally randomly integrated into the mouse genome (commonly within non-coding DNA, although gene disruption via the inserted transgene has sometimes been known to occur). The forward and reverse primers are thus designed within the transgene and a simple PCR is used to merely determine if the transgene is present or absent within the mouse. If preferred, the copy number of the inserted transgene can be determined with Southern blot analysis (Cho et al.,2009 and Haruyama et al.,2009), although a quantitative PCR assay can also be used. Thermal asymmetric interlaced (TAIL)-PCR can also be performed on a transgenic mouse line to identify the transgene integration site (Liu and Chen 2007, Pillai et al. 2007).

We frequently use MacVector, or the NCBI genome browser (see Method 2, and internet resources) to identify pairs of primers to amplify various gene segments in both the wild type gene and the mutant gene.

Method 1: Identification of primer sequences using MacVector

1. Paste the sequence about the mutated allele or transgene into the empty nucleotide entry form of the MacVector software.

2. Select analysis in the tool bar.

3. Select primer design/test pairs from the drop-down menu

If a sense and anti-sense primer pair are known, the pairs can be tested in silico to determine whether the primers should generate an amplicon as well as to identify the optimal primer annealing temperature. This temperature is critical for initial binding of the primer template to the double stranded DNA as well as for each cycle of the PCR reaction. If a sense and anti-sense primers are not known, then one can use the Find 5’ and Find 3’ radio buttons to identify primer candidates based upon certain factors such as Primer length, GC content, and potential for self-complementarity. The software may find one or more product sizes based upon the sequence range and search criteria. The output is depicted graphically as Product 1 through Product N. The Primer3 report shows the input primer sequence, the primer binding site and the approximate melting temperature (see Figure 3).

Method 2: Identification of primer sequences using NCBI- Primer BLAST

1. Go to Primer BLAST (see internet resources) and input your PCR template sequence.

   Several search criteria are available in each data box. Generally, the most critical entry panel is the desired PCR product size (typically between 400 base pair and 800 base pair amplicons). Other parameters such as minimum and maximum melting temperature can be selected. Usually, the default settings for melting temperatures have been optimized.

2. Select the blue “Get primers” tab.

The website will process the request. The computation time to complete the request will vary depending upon the number of other requests. When completed, the output will be detailed Primer pair reports including the primer sequences (5’ to 3’), amplicon length, calculated melting temperature, and an assessment of self-complementarity (see Figure 4).

Figure 4. (A,B,C):

A screenshot of Primer-Blast website showing different entry fields to retrieve pairs of primer designs. Any accession numbers or direct sequences can be entered in the template section. Note that the size of the amplicon section highlighted showing 800–1,000 bp range, the numbers in the primer melting temperature section is showing default numbers. B: Screen shot showing specificity by checking parameters such as Database and Origin. These sections should be filled out to accordingly to retrieve efficient primer sets. C: Schematic diagram of primer design Nuclease assay. Pr1 and Pr2 amplifies a total of 800 bp. The red thunder arrow shows the site of double strand break. In the gel picture, sample #2 demonstrates the result of small indels thus cleavage of heterogenous allele by T7 Nuclease, whereas sample #3 represents deletion of one allele. Sample #4 shows a case where there are more than three alleles which are wild-type size amplicon, deletion, and cleavage by T7 Nuclease. Sample #6 is amplicon from wild type sample (C57BL/6Tac) and M stands for DNA ladder (1kb plus DNA ladder from NEB).

Further information about the different types of genetically engineered mutations and the protocols needed for genotyping of each mutation are described below.

Method 3: Typical endpoint PCR reactions

Materials

Basic 96-well thermal cycler capable of short ramp times between cycling temperatures (e.g., VeritiPro Thermal cycler-96 well, cat. no. A48141, ThermoFisher Scientific), or equivalent

Thin-walled DNase/RNase-free reaction tubes (0.2 ml) suitable for temperatures exceeding 95°C, (Fisher Scientific, cat. no. 14–230-228) or equivalent

Pipettors (e.g., Gilson PIPETMAN classic, Fisher Scientific) or equivalent:

200–1000 μL (cat. no. F123602G)

20– 200 μL (cat. no. F123601G)

2–20 μL (cat. no. F123600G)

1–10 μL (cat. no. F144802G)

Disposable, DNase-free pipette tips containing aerosol barrier filters that fit the pipettes listed above (e.g., Thermo Scientific ART Barrier Pipette Tips through Fisher Scientific), or equivalent

200–1000 μL (cat. no. 21–236-2A)

20–200 μL (cat. no. 21–236-1)

2–20 μL (cat. no. 21–236-4)

1–10 μL (cat. no. 21–236-5)

Clear Microcentrifuge tubes, 1.7 ml (DNAse/RNase free), (Crystalgen, cat. no. L-2052) or equivalent

Ex Taq DNA polymerase (TakaraBio, cat. no. RR001A): contains Ex Taq polymerase, 10X Ex Taq buffer, dNTP mix

Molecular biology grade water (Quality Biological, cat. no. 351–029-721) or equivalent

Custom oligonucleotide primers (e.g., eurofinsgenomics.com or idtdna.com):

15–40 nucleotides, salt-free, 25 nmol synthesis scale

The typical process is to create a master mix that contains all the components listed below for a 50 μL reaction. The volume of each component is then multiplied by the number of samples to analyze plus one or two reactions to account for pipetting errors.

10X ExTaq buffer:

(5.0 μL per 50 μL reaction). This buffer (included with the Ex Taq DNA-polymerase) contains an optimized concentration of Mg²⁺ and KCl at a ten times concentrated solution. If needed, this buffer can also be purchased without Mg²⁺and can be added separately to the supplied buffer. In general, additional Mg²⁺ improves the generation of the final PCR product. However, too much Mg²⁺can also reduce specificity of the Taq polymerase.

dNTP solution:

(4.0 μL per 50 μL reaction). This solution is a mixture of all four dinucleotide phosphates (dATP, dCTP, dTTP, and dGTP). The stock concentration is 2.5 mM, with the final concentration 200 μM of each dinucleotide phosphate base.

Two or three oligonucleotide primers:

(1.0 μL of a 10 μM solution of each primer per 50 μL reaction). In this example the final concentration is 0.2 μM of each primer. The final concentration may vary between 0.2 μM and 1.0 μM and must be determined empirically. Primers are often shipped lyophilized. Perform a pulse spin before opening the tube and dilute primers to a 100 μM stock solution (for every 1 nanomole add 10 μL of PCR-grade water or TE). Each primer is then diluted to a 10 μM working stock through a 1:10 dilution using DNase-free water.

Template DNA:

See c (Basic Protocols 1–4), typically a 100 nanogram/μL concentration of DNA is sufficient for routine PCR. We generally use 4 μL (200 nanogram). PCR amplification can still occur even with a lower concentration of approximately 20 nanogram/μL.

ExTaq DNA polymerase:

Typically, 0.25 μL for a 50 μL reaction is sufficient. The stock polymerase is 5 units/μL. The final concentration is therefore 0.025 units/μL. The optimal concentration must also be empirically determined.

DNase-free water (Molecular Biology grade):

The remainder volume is brought up to 50 μL for one reaction.

A commonly used PCR needed for genetically engineered conditional knockouts using Cre/loxP technology is for amplification of the Cre gene (Sreenath et al., 2003) for a 50 μL reaction is shown in Table 3 (A,B).

Table 3 (A,B):

PCR protocol and thermal cycler profile to detect the Cre gene

Example of a PCR reaction master mix to amplify the Cre gene

Primer#1: 5’- GCCTGCATTACCGGTCGATGCAACGA-3’	Primer#2:5’- GTGGCAGATGGCGCGGCAACACCATT-3’
Reaction volumes for 1 sample
Nuclease-free water	34.75 μl
2.5 mM dNTP solution	4.0 μl
10X ExTaq buffer solution	5.0 μl
Primer #1 (10 μM)	1.0 μl
Primer #2 10 μM)	1.0 μl
ExTaq DNA polymerase (5U/μl)	0.25 μl
Genomic DNA (~200 nanogram)	4.0 μl
TOTAL VOLUME	50 μl

Thermal cycler program to amplify the Cre gene

1. Initial Denaturation at 94°C	3 minutes
2. Denaturation at 94°C	45 seconds
3. Annealing primers at 67°C	30 seconds
4. Extension of the amplicon at 72°C	45 seconds
5. Repeat steps 2. To 4. For 30 cycles
6. Final extension of the amplicon at 72°C	10 minutes
7. Ramp temperature to 4°C	~30 minutes to 1 hour

1. Thaw the 10X ExTaq buffer, dNTP solution, and primers on wet ice.

   ExTaq polymerase is in a glycerol solution and should be placed in wet ice

   In one microcentrifuge tube per PCR reaction, add the appropriate volume of water.

   Increase the volume of each component in the master mix above depending upon the number of samples to be amplified.

2. Next, add the 10X PCR buffer

3. Add the dNTP solution

4. Add each primer

5. Add the ExTaq polymerase last

6. Mix the solution well and dispense into each 0.2 ml PCR reaction tubes (typically 46 μL)

7. Add template DNA (typically 4 μL, or approximately 100 −200 ng/μl)

8. Place each PCR reaction tube in the thermal cycler

The amplification program is described below

1. Activation: The first phase involves an activation step, usually between 94°C to 95°C that is held for between 2–5 minutes for a standard PCR reaction (usually longer for Hot start PCR).

2. Cycling parameters: The subsequent steps are repeated generally between 30 and 40 cycles depending upon the amount of starting template.

   1. Denaturation: 30–45 seconds at 94°C to denature and separate the DNA strands.
   2. Annealing: The initial temperature is lowered to between 50°C and 65°C to allow the oligonucleotide primers to bind to each strand of the template DNA. The annealing temperature is usually 5°C below the primer melting temperature. The melting temperature is the threshold temperature for the primer to either bind or stay unbound to each template strand and is affected by the number of GC content and the salt concentration. The annealing temperature is usually indicated in the output from Method 1 or Method 2 above. The optimal annealing temperature should be determined empirically. Annealing time is typically 30–60 seconds.
   3. Extension: The temperature is raised to between 68°C and 72°C to allow the DNA polymerase to extend the nascent strand using the free dinucleotide phosphate bases in the dNTP mix. In general, 30 seconds per 500 base pair amplicons.

3. Final extension: After the cycling steps are completed, a final extension step at 72°C is added to complete the generation of the amplicon. The final extension time usually varies from 2 minutes to as much as 10 minutes depending upon the amplicon size.

4. Hold step: The final step is usually to bring the reaction temperature to approximately room temperature, or 4°C for further processing (e.g., agarose gel electrophoresis) to identify the expected amplicon size or sizes.

The size of the amplification product should be 726 base pairs for any mouse genomic DNA bearing the Cre modification.

An example of a PCR reaction to amplify a PCR product from the mouse beta-globin gene based upon Konkel et al.,1978 is shown in Table 4 (A,B).:

Table 4(A,B):

Example of a PCR reaction master mix to amplify the mouse beta-globin gene

Primer#1: 5’- CCAATCTGCTCACACAGGATAGAGAGGGCAGG-3’	Primer#2:5’- CCTTGAGGCTGTCCAAGTGATTCAGGCCATCG-3’
Reaction volumes for 1 sample
Nuclease-free water	34.75 μl
2.5 mM dNTP solution	4.0 μl
10X ExTaq buffer solution	5.0 μl
Primer #1 (10 μM)	1.0 μl
Primer #2 10 μM)	1.0 μl
ExTaq DNA polymerase (5U/μl)	0.25 μl
Genomic DNA (~200 nanogram)	4.0 μl
TOTAL VOLUME	50 μl

Thermal cycler program to amplify the mouse beta-globin gene

1. Initial Denaturation at 94°C	3 minutes
2. Denaturation at 94°C	30 seconds
3. Annealing primers at 60°C	90 seconds
4. Extension of the amplicon at 72°C	2 minutes
5. Repeat steps 2. To 4. For 35 cycles
6. Final extension of the amplicon at 72°C	10 minutes
7. Ramp temperature to 4°C	~30 minutes to 1 hour

The size of the amplification product should be 494 base pairs for any mouse genomic DNA. This profile is useful as a control for the quality of genomic DNA, and for the PCR reagent master mix (see also troubleshooting). The amount of template genomic DNA along with the number of cycles may need to be optimized. Around 30–45 cycles are needed with most PCR reactions. Annealing temperature may also need to be adjusted but first, one can begin with 5°C below the primer melting temperature.

Occasionally, some PCR protocols for genotyping will differ from the standard PCR protocol listed above. One strategy is to use “Touchdown PCR” to generate the expected amplicon (Green-MR et al., 2018). In this method the annealing temperature starts out at a higher temperature (and stringency of primer binding to the template strands) than the calculated annealing temperature and gets gradually lowered at each subsequent cycle. For example, during the first 10 cycles, the annealing temperature is slightly above the calculated temperature. Then during the subsequent 20–25 cycles, the temperature is lowered by 0.5°C to 1°C at each cycle. This generates an initial “high stringency” number of copies. The subsequent cycles build upon these initial copies during the exponential phase of the amplification. The touchdown PCR method is used when there is not an exact match between primer and template, or when there is variability between strains of mice for the particular gene segment to be amplified. In one example, the genotyping for a dual fluorescent Cre reporter mouse (mT/mG mice, #007676 from the Jackson Laboratory repository (see internet resources), (Muzumdar et al., 2007) involves the annealing temperature being set first at 65°C and gradually lowered by 0.5°C per cycle for 10 cycles. Then the annealing temperature is set at 60°C for the remaining 28 cycles (no ramp down of the temperature). The touchdown method is used because of sequence variation in one of the three primers used in this protocol and due to differences in the optimal annealing temperature of each primer.

Identification of wild-type, heterozygote, and knock-out genotypes

Before CRISPR technology transformed the gene editing process, gene knockout mice were generated through the manipulation of mouse embryonic stem cells (mES cells, see review in Goldstein, 2001). In this strategy, two flanking regions of identical DNA to the gene of interest surround a drug resistance cassette. This segment of DNA is electroporated into ES cells to then recombine and replace the coding portion of the native (wild type) gene. This occurs very infrequently through natural homologous recombination events. The drug resistance cassette acts as a selection marker in that the ES cells are cultured in vitro in a medium containing the same drug. Any ES cells that do not possess the drug resistance cassette will die off, leaving the ES cell with targeted gene modification to persist and develop clonal populations. These modified ES cells are then microinjected into early-stage mouse embryos to generate chimeric mice (mice that possess organs containing gene edited cells and wild-type cells, see review Limaye-A et al.,2009). Through eventual breeding, the line can be made into a heterozygous (one edited allele) and one wild-type state, or homozygous where both alleles have been edited.

It is often useful to be able to separate these three genotypes, particularly if the phenotype is subtle. The strategy relies upon identification of a common primer sequence binding to a region outside of the modification (e.g., drug resistance cassette) and a second specific primer binding within the modification. A third unique primer is generated that binds to the wild-type sequence that was replaced by the modified sequence. Therefore, after PCR amplification, if both alleles are wild-type, one PCR product is generated. If both alleles have been modified, then a different amplicon is generated. If one allele is modified and the second is wild type, then both amplicons are generated and can be separated following gel electrophoresis (see Figure 5). The two PCR products need to be of sufficient different base pair lengths to identify the amplicon from each allele on an agarose gel. All three primer solutions can be combined in the same PCR reaction provided that the annealing temperature is approximately the same.

Figure 5:

PCR strategy to identify and distinguish wild-type, heterozygous, and homozygous genotypes when there is a selectable marker. A common “forward” primer is depicted in red. A specific wild-type “reverse” primer is depicted in green. A selection marker specific “reverse” primer is depicted in purple. Only when there is the presence of modified sequence will you get an amplification product of the common forward primer (red) and specific reverse primer (purple).

Before proceeding to create a new knockout mouse using gene editing tools like CRISPR, one may want to first determine if there are KOMP (Austin et al., 2004) repository mice or ES cells already available, such as through the Mutant Mouse Resource & Research Center (MMRRC) (see internet resources) or with the Jackson Laboratory (see internet resources). Of note, mutations in mice may lead to embryonic or perinatal lethality. In certain cases, it may be of interest to have a PCR technique that will allow the investigator to learn the gender of the mutant mice. Sex genotyping can be done using SRY oligos (Kunieda et al., 1992) or a simplex PCR based on the X and Y chromosome-specific genes Jarid1c and Jarid1d (Clapcote and Roder, 2005).

Genotyping of CRISPR gene edited mice

Introduced in 2012, the CRISPR gene editing system has become the method of choice to generate genetically modified cell lines or genetically modified mouse models. The first step in CRISPR-based gene editing is to induce double-strand breaks (DSBs) on a target region by a guide RNA forming a complex with endonuclease Sp (Streptococcus pyogenes) Cas9 (Horvath et al., 2010; Cong et al., 2013; Aida et al., 2014; Mashimo, 2014). Then, by the non-homologous end joining (NHEJ) repair pathway, the error prone repair pathway triggered by the cell’s own system, will either insert or delete random nucleotides from the DSB site, called indels (Cong et al., 2013; Aida et al., 2014). These random indels by CRISPR have been useful to disrupt the gene and generate a non-functional coding region, generally by the creation of a frameshift mutation (Tuladhar et al., 2019). Or, a precise mutation can be achieved by the Homologous Directed Repair (HDR) pathway, an alternative to naturally occurring NHEJ repair mechanism initiated by the DSB. In the case of the HDR repair pathway, a repair cassette which includes the desired insertion or modification, flanked by segments of DNA homologous to the blunt ends of the cleaved DNA. is added to the guide RNA/Cas9 complex. Therefore, these donor templates will be inserted upon the generation of the DSB resulting in a precise, desired mutation (Kim et al., 2014).

Although the principle of genotyping CRISPR gene edited animals is the same as that of traditional transgenic animals, it is also different to some extent. First, the founder animals could display bi-allelic or even multi-allelic gene editing due to the persistence of cutting by the CRISPR complex beyond the two-cell stage (Gurumurthy et al., 2019). Secondly, due to the presence of similar protospacer regions in the genome, off target edits can occur. Several tools are available that identify the region where off-target cuts are more likely to occur especially if these off-target hits are in exon regions. The list of off target genes can be checked for edits using a PCR strategy described below. Or, for the most thorough off-target assessment, whole genome sequencing may be performed. Lastly, efficient, and stepwise strategic genotyping protocols are necessary since there can be an abundance of positive animals in one round of CRISPR editing. To this end, the purpose of the current chapter is to demonstrate efficient genotyping strategies for various types of gene editing by CRISPR.

Basic Protocol 6: T7E1/Surveyor assays to detect indels (insertions or deletions) following CRISPR editing

T7 Endonuclease, originating from Escherichia coli bacteriophage, is a junction-resolving enzyme, that selectively binds and cleaves Holliday junctions with high specificity for branched structures in double-stranded DNA, such as cruciform DNA, but also it has a strong preference for cutting single-stranded DNA (Melvin et al., 1970; Guo et al., 2014). The T7E1/Surveyor assay begins by generating an amplicon that covers the region where a potential indel occurred. If an indel occurred, there will be a mismatch or mismatches in the sequence of the PCR amplicon. The T7E1/Surveyor enzyme is then added, and the mismatch is resolved by the creation of a double strand break. This break can be identified through gel electrophoresis by comparing the separated fragments to a “wild-type” amplicon where no fragments would be expected. The T7/E1 Surveyor assay has been used as one of the most convenient and informative assays to screen CRISPR gene edits. While the assay does not offer a more thorough result compared to DNA sequencing, it can provide a speedy screening method to narrow down and identify specific positive (founder) CRISPR animals efficiently. Furthermore, this assay is suitable for screening blastocysts that are cultured after CRISPR editing as a method of evaluating the double stranded break (DSB) efficiency of each guide RNA.

Materials

Alcohol Swab (Covidien, cat. no. 6818)

Ring handle ear punch, 5mm (Roboz Surgical Instrument Co., cat. no. 65–9902)

Alcohol resistant marker pen, black (Fisher brand, cat. no. 1400–20-FSC)

DNA lysis buffer (See reagents and solutions):

Tris HCl, pH 8.0 (Thermo Scientific, cat. no. AM9855G)

EDTA (Millipore-Sigma, cat. no. E7889–100ML)

Sodium Chloride (Millipore-Sigma, cat. no. S5886)

Sodium dodecyl sulfate (Millipore-Sigma, cat. no. L3771)

Proteinase K (Thermo Scientific, cat. no. 25530–049)

Isopropyl Alcohol (Millipore-Sigma, cat. no. I9516)

Absolute Alcohol (Millipore-Sigma, cat. no. 459844

GOtaq Green Master Mix (Promega, cat. no. M7828)

PrimeStar Max (Clontech, cat. no. R040A)

Nuclease-free water (Millipore-Sigma, cat. no. W4502–50ML)

Custom oligonucleotide primers (e.g., eurofinsgenomics.com or idtdna.com; Lyophilized, see reagents and recipe for generating 10 pmol primer)

2% Agarose gel (See reagents and solutions)

Agarose (MP Biomedicals, cat. no. 11AGAH0250)

Ethidium Bromide solution (Alfa Aesar, cat. no. J63382)

CAUTION: Ethidium Bromide is a known mutagen. PPE must be used, and any downstream waste products must be handled according to institutional guidelines.

50x TAE buffer (Thermo Scientific, cat. no. B49)

GeneArt^™ Genomic Cleavage Detection System (Thermo Scientific, cat. no. A24372)

Smart kit E4 LTS for 20, 100, 1000 μl electronic pipette (Rainin, cat. no. 30386731) or equivalent

Multi-Channel pipette (Rainin, cat. no. pipette Lite XLS 17013803) or equivalent

Mastercycler, nexus gradient (Eppendorf, cat. no. 6331) or equivalent

Refrigerated Centrifuge (Eppendorf, cat. no. 5427R) or equivalent

Vortex mixer (Daigger, cat. no. Vortex-Genie2) or equivalent

AirClean 600 PCR workstation (AirClean Systems cat. no. AC624), or equivalent

Thermo Scientific^™ Owl^™ A2 Large Gel Systems (ThermoFisher, cat. no. 09–528-102)

Microwell comb (ThermoFisher, cat. no. A2-MT2C)

Imaging system (Azure Biosystems, Azure 300)

Microwave (Panasonic, cat. no. S28645) or equivalent

Heat block (Eppendorf, Thermomixer F1.5) or equivalent)

Magnetic Stirrer (Fisher Scientific, cat. no. S88857200) or equivalent

PCR Tube Strips, 0.2ml (Eppendorf, cat. no. 951010022) or equivalent

PCR plate, semi skirted (Eppendorf, cat. no. 951020346) or equivalent

1.5ml microtubes (Ambion, cat. no. AM12400 )or equivalent

Latex or Nitrile gloves (Fisher Brand, cat. no. 11–394-15 or 19–041-171) or equivalent

1000 μl BioClean Ultra tips (Rainin, cat. no. 30389211) or equivalent

200μl BioClean Ultra tips (Rainin, cat. no. 17005859) or equivalent

20μl BioClean Ultra tips (cat. no. 17005860) or equivalent

S-Blocks and Round-Well Blocks (Qiagen, cat. no. 19585) or equivalent

AirPore Tape Sheets (Qiagen, cat. no. 19571) or equivalent

Magnetic Bar (Fisher Scientific, cat. no. 14–513-68) or equivalent

500ml flask (Fisher Scientific, cat. no. 41030250) or equivalent

1. Copy and paste about 1,500 bp of genomic sequence spanning the target region in the PCR Template section.

2. At the primer parameter section, set up your amplicon size range from 800 to 1,000bp, enter 5 or 10 for number of primers then use a melting temperature of 60°C with a maximum Tm difference of 3 (Figure 4A).

   No action is necessary on Exon/intron selection as this section is for an mRNA template.

3. Choose Genomes for selected organism in the Primer Pair Specificity Checking Parameters section and enter 4000 bp for the maximum target amplicon size (Figure 4B).

When choosing the primer pairs at the result screen, it is ideal to have suitable size gap between T7/E1 nuclease cleaved bands for a clear detection in the gel image analysis. Otherwise, it would be difficult to resolve two bands of similar sizes. Therefore, a primer pair that will place the target site at the 1/3 of total PCR amplicon size should be chosen. For example, with the PCR product size of 800 bp, the location of the target site can be apart, resulting in PCR bands of 550 and 250 bp size (Figure 4C).

Preparing DNA Lysates & Extraction of DNA from the ear pinna

Typically, when performing ear punch collection, an ear tag is placed in one side of pinna for identification purposes then an approximate 5mm-radius pinna biopsy sample can be obtained by applying the Ring handle ear punch to the other side of the mouse ear. Once the pinna sample is obtained, a 1.5ml microtube should be labeled with the matching identification ear tag number using an alcohol resistant pen. At this point, the tubes can be stored at −20°C for later purification. The following are detailed technical steps for DNA extraction by alcohol precipitation.

1. Add 500 μl of the DNA lysis buffer (see reagent and recipe) and then place the tube in the heat block which is set at 57°C.

2. After 2 hours of incubation, check the tube for any debris by tapping the tube make sure all the tissue is well dissolved. If there are any remaining small tissue debris, place the tube back in the heat block until the debris is absent.

3. Once the DNA lysate is cooled down to room temperature, add 500 μl of isopropanol to the ear punch lysate. The sample is then mixed by inverting the 1.5ml tubes several times-do not apply a vortex to the tube. Centrifuge for 10 minutes at 15,000 X g, 4°C.

   Remove the tube from the centrifuge carefully, do not agitate the precipitated DNA that has formed at the bottom side of the tube. DNA precipitation should be easily visible due to the small hair and tissue debris, which has no adverse effect on PCR reactions.

4. Carefully drain away the supernatant from the microtube.

5. Add 500 μl of 70% alcohol to wash the crude pellet, then centrifuge the tubes for 5 minutes at 15,000 X g, 4°C.

6. Finally, carefully drain the alcohol so that the precipitated DNA is not disturbed. The pellet in the microtube tube is then air dried by inverting the tube on the clean paper towel for 5 minutes.

7. Once all the alcohol has evaporated, add 250μl of Nuclease free water and dissolve the pellet by tapping the tube several times and briefly vortex.

The DNA sample for PCR amplification can be stored at 4°C for more than 1 year.

PCR Reactions

It is crucial to set up PCR reactions in a manner that does not amplify non-specific bands or generate an unexpected positive band in a negative control sample (most of the time, the negative DNA control should be from a wild type animal). The following steps are recommended for this procedure to reduce the chances of cross-contamination between samples and should be performed inside a chamber that minimizes air flow such as the Airclean 600 PCR workstation.

1. Transfer all the reagents needed for PCR reactions including PCR master mix, primers, and DNA template to a chilled rack or ice bucket containing wet ice inside the PCR workstation. If any of the reagents are frozen, place those tubes at room temperature until completely thawed then place back into the chilled rack or ice bucket until ready.

2. Aliquot 50 μl of prepared genomic DNAs into PCR strip tubes or PCR plates in the order of identification number along with negative and positive controls at the end.

   Aliquoting DNA samples into PCR strip or plate will enable one to use a multi-channel pipette for the next steps, thus reducing the error due to sample omission or duplication. Therefore, it is highly recommended to use a multi-channel pipette in this assay.

3. Prepare a PCR Master mix by adding the 2X Taq polymerase, primers, and water in a tube (see example in Table 2). Briefly vortex the tube. Add the 19 μl of PCR master mix solution to a new PCR tube in a strip or plate, depends on the number of total samples in the reaction.

   We recommend using pipettes with an aliquoting function to reduce cross contamination.

4. Add 1 μl of DNA sample to each PCR strip or plate in the correct order using a multichannel pipette.

5. Carefully place adhesive film on the top of the PCR plate or affix the top of PCR strip. The PCR strip or plate can then be spun down briefly before being placed into the PCR cycler.

6. Start a PCR cycler with the appropriate program.

Table 2.

Composition of PCR mix

Reagent	Volume
Nuclease Free Water	7μl
2x GO taq mix	10μl
Primer1 (10pmol)	1μl
Primer2 (10pmol)	1μl
DNA (~10–100 ng)	1μl
Total	20μl

A typical PCR reaction program is shown in Table 5.

Table 5.

PCR reaction cycle

Step	Temperature	Incubation Time
Step1	95°C	5mins
Step2, 34 cycles	95°C 60°C 72°C	45 secs 1 min per kb 45 secs
Step3	72°C	4mins
Step4	4°C	∞

Agarose Gel electrophoresis to verify the PCR product

Before performing T7E1 nuclease assay, the PCR product should be subjected to gel electrophoresis to ensure a specific single band.

To prepare for agarose gel electrophoresis, refer to the reagents and recipe section for detail.

1. To confirm the correct size and roughly estimate the amount of the amplicon, load 3 μl of PCR product onto the 2% agarose gel along with the 1kb plus DNA ladder at the end of the well row.

2. Run the gel electrophoresis at 80 V/10 cm for 30 minutes then place the gel onto UV imager tray to take a digital image of the gel.

3. Once a single and clear PCR amplification band is confirmed with control DNA, proceed to the T7E1 nuclease incubation.

Adjust any conditions such as annealing temperature, extension time, and the amount of DNA if there is either a non-specific band or no amplification product detected.

T7E1 nuclease incubation (Basic Protocol 6)

The first step of T7E1 nuclease treatment is to denature and then randomly anneal the PCR product to form heterogeneous DNA duplexes with or without indels.

1. Before the denaturing process, 5 μl of PCR product is combined with 1.5 μl of Detection buffer and 7.5 μl of nuclease free water in a 0.2 ml PCR tube. We recommend making a master mix (Table 6) of the detection buffer and water, then add 9 μl to each PCR tube. Then add 5 μl of PCR product to the PCR tubes using multi-channel pipette.

   It is important that the maximum volume of PCR product is not over 50% of the total volume of the reaction. Higher volume of PCR buffer will negatively affect the efficiency of the T7 endonuclease). While most PCR buffers are compatible with this assay, we highly recommend testing the PCR buffer to be used before performing the actual reaction.

2. Incubate the samples at 95°C for 10 minutes in the PCR cycler to separate the double stranded DNA.

3. The sample is re-natured by a slow cool down process. If the lab is equipped with PCR cycler that is capable of ramping time of 0.1°C/second, a PCR program can be used for this step (Table 7). Alternatively, the PCR tubes can be kept in the PCR cycler block to cool for 30 minutes immediately after 10 minutes of denaturation.

   Once the samples are re-natured, the samples can be stored at −20°C.

4. Briefly spin down the samples to remove water condensation caused by the slow cool procedure.

5. Aliquot 4 μl of the reaction to a new PCR strips/plate and save the product for later gel electrophoresis as a negative control for each sample.

6. Following the brief spin down, 1 μl of T7 nuclease enzyme is added to each PCR tube and incubated at 37°C for 1 hour.

Table 6.

Sample preparation for Denaturation/Re-naturation process

Reagent	Volume/Sample	Volume For Master Mix
PCR product	5μl	NA
10x Detection Reaction Buffer	1.5μl	1.5 x (n+1)μl
Nuclease Free Water	7.5μl	7.5 x (n+1)μl
Total	14μl	NA

Table 7.

A PCR program for denaturation procedure

Step	Temperature	Time	Ramping Speed
1	95°C	5min	NA
2	95°C-85°C	NA	-2°C/sec
3	85°C-25°C	NA	-0.1°C/sec
4	4°C	∞	NA

Agarose Gel electrophoresis and analysis

Immediately after the reaction, the samples should be loaded onto a 2% Agarose gel along with a defined fragment standard such as 1kb DNA ladder to analyze the potential indel. To prepare the agarose gel and set up the gel tank for electrophoresis with running buffer, refer the reagents and recipe section for details. To detect if the T7E1 mediated digest occurred between PCR products with and without T7E1 nuclease treatment, the samples should be loaded side by side (Figure 5C).

1. Load the 4 μl of PCR product without T7E1 nuclease treatment first using multi-channel pipette into micro well, filling every other well of the gel first.

2. Load the PCR product with the T7E1 nuclease treatment onto the wells right next to each other with matching duplicate samples.

3. Once all the samples and DNA ladder are loaded, apply the electric current at a voltage of 80V/cm for 30 minutes.

4. The agarose gel can then be imaged using a gel imaging system to determine the relative proportion of DNA contained in each band using desired gel analysis software.

Basic Protocol 7: Detection of off-target mutations

Off-target mutations can occur due to the presence of similar, but not exact protospacer and PAM sequences throughout the genome. If these off-target mutations are observed at frequencies greater than the intended (on-target) mutation, this may lead to genome instability and disrupt the functionality of otherwise normal genes, which can be a major complication whenever using the CRISPR/Cas9 system in biomedical and clinical applications.

Although the targeting specificity of Cas9 is believed to be tightly controlled by the 20-nt protospacer sequence of the sgRNA and the presence of a PAM adjacent to the target sequence in the genome, potential off-target cleavage activity could still occur in DNA sequences with even three to five base pair mismatches in the PAM-distal part of the protospacer sequence (often referred to as the non-seed sequence). Moreover, previous studies have demonstrated that different guide RNA structures can affect the cleavage of on-target and off-target sites. Crystal structure studies and single-molecule DNA curtain experiments suggest that while the PAM site is essential for the initiation of Cas9 binding, the seed sequence corresponding to 3′ end of the crRNA complementary recognition sequence, directly adjacent to PAM, is also critical for subsequent Cas9 binding, R-loop formation, and activation of nuclease activities in Cas9.

Materials

Alcohol Swab (Covidien, cat# 6818)

Ring handle ear punch, 5mm (Roboz Surgical Instrument Co., cat# 65–9902)

Alcohol resistant marker pen, black (Fisher brand, cat# 1400–20-FSC)

DNA lysis buffer (See reagents and solutions):

Tris HCl, pH 8.0 (Invitrogen, cat. no. AM9855G)

EDTA (MilliporeSigma, cat. no. E7889–100ML)

Sodium Chloride (MilliporeSigma, cat. no. S5886)

Sodium dodecyl sulfate (MilliporeSigma, cat. no. L3771)

Proteinase K (Thermo Scientific, cat. no. 25530–049)

Isopropyl Alcohol (MilliporeSigma, cat. no. I9516)

Absolute Alcohol (MilliporeSigma, cat. no. 459844)

PrimeStar Max polymerase (Clontech, cat. no. R040A)

Nuclease-free water (MilliporeSigma, cat. no. W4502–50ML)

Custom oligonucleotide primers (e.g., eurofinsgenomics.com or idtdna.com; Lyophilized, see reagents and recipe for generating 10 pmol primer)

2% Agarose gel (See reagents and solutions)

Agarose (MP Biomedicals, cat. no. 11AGAH0250)

Ethidium Bromide solution (Alfa Asra, cat. no. J63382)

CAUTION: Ethidium Bromide is a known mutagen. PPE must be used and any downstream waste products must be handled according to institutional guidelines.

50x TAE buffer (Thermo Scientific, cat. no. B49)

NucleoSpin Gel and PCR cleanup (Clontech, cat. no. 740609)

Smart kit E4 LTS for 20, 100, 1000 μl electronic pipette (Rainin, cat. no. 30386731) or equivalent

Multi-Channel pipette (Rainin, cat. no. pipette Lite XLS 17013803) or equivalent

Mastercycler, nexus gradient (Eppendorf, cat. no. 6331) or equivalent

Refrigerated Centrifuge (Eppendorf, cat. no. 5427R) or equivalent

Vortex mixer (Daigger, cat. no. Vortex-Genie2) or equivalent

Thermo Scientific^™ Owl^™ A2 Large Gel Systems (ThermoFisher, cat. no. 09–528-102

Microwell comb (ThermoFisher, cat. no. A2-MT2C)

Imaging system (Azure Biosystems, Azure 300)

Microwave (Panasonic, cat. no. S28645) or equivalent

Heat block (Eppendorf, Thermomixer F1.5) or equivalent

Magnetic Stirrer (Fisher Scientific, cat. no. S88857200) or equivalent

PCR Tube Strips, 0.2ml (Eppendorf, cat. no. 951010022) or equivalent

PCR plate semi skirted (Eppendorf, cat. no. 951020346) or equivalent

1.5ml microtubes (Ambion, cat. no. AM12400) or equivalent

Latex or Nitrile gloves (Fisher Brand, cat# 11–394-15 or 19–041-171) or equivalent

1000 μl BioClean Ultra tips (Rainin, cat. no. 30389211) or equivalent

200μl BioClean Ultra tips (Rainin, cat. no. 17005859) or equivalent

20μl BioClean Ultra tips (cat. no. 17005860) or equivalent

S-Blocks and Round-Well Blocks (Qiagen, cat. no. 19585) or equivalent

AirPore Tape Sheets (Qiagen, cat. no. 19571) or equivalent

Magnetic Bar (Fisher Scientific, cat. no. 14–513-68) or equivalent

500ml flask (Fisher Scientific, cat. no. 41030250) or equivalent

NucleoSpin^® Gel and PCR Clean-up kits (Clontech, cat. no. 740609)

Nanodrop 2000 (Thermo Scientific, cat. no. ND2000CLAPTOP)

Generating a list of Linked Off-Targets

The first factor for designing sgRNA for a CRISPR editing project, is the specificity of certain guide RNA within the whole mouse genome. This is called a guide specificity score, which is ranked on a scale from 1–100 with higher numbers representing fewer number of off target genes. One useful tool for CRISPR gene editing is CRISPOR (see internet resources) which is used in the following section (Concordet and Haeussler, 2018).

1. On the first screen, paste the target genomic sequence then choose genome database and different types of Cas9s (Fig 6A). Typically, we use the default of Streptococcus Pyogenes (Sp Cas9).

   Then the list of sgRNAs will be lined up as a table with the higher MIT score listed first. Also displayed, are other factors such as efficiency, and finally displayed are the list of off targets (Fig 6B).

2. Select “chr” so only the off-target genes located in the same chromosome will remain. The “exon” can also be checked if only the off-targets located in exon of the genes will be considered.

3. Download the primer pairs for the list of the off-target genes by clicking the list of “starter genes” to synthesize the appropriate primer sequences.

Figure 6. (A,B):

An initial screenshot of CRISPOR website showing first 3 steps to design guide RNAs for CRISPR gene editing. In Step 1, copy and paste the DNA sequence from region of interest. Select mouse genome database at Step2 and choose your choice of Cas9 (Streptococcus pyogenes Cas9 (SpCas9), shown at the Figure 6A is the most widely used Cas9 enzyme). B: The result screen after submitting the sequence and parameters. The list of gRNAs is shown from the highest MIT score to the lowest. To choose the best gRNAs, narrow the off-target list by clicking exon and chr check box (black arrows).

Preparing Samples for DNA Sequencing

For DNA extraction, PCR reaction and gel loading/analysis, please refer to Basic Protocol 4. The primers, enzymes and dNTPs in the reaction should be removed for best sequencing results. This can be achieved by column purification (NucleoSpin^® Gel and PCR Clean-up kits), gel purification, or enzymatic treatment with Exonuclease I. The following procedure is for column purification using a commercial kit. Note that sequencing of PCR product requires at least 50 ng/μl. Thus, to collect a suitable amount of DNA, scale up the volume of each PCR reaction to 100 μl.

1. Add 2 volumes of buffer NTI (provided in the NucleoSpin clean up kit) to the 1 volume of PCR product in the microtube and mix well by pipetting up and down slowly. For example, add 400 μl of NTI buffer to 200 μl of the PCR product.

   Depending on the initial PCR volume, this procedure can be repeated since the capacity of the column is 600 μl.

2. Load the NTI + PCR product mixture into the column. Attach a 2 ml collection tube to the bottom of the column, spin at 11,000 X g for 30 seconds.

3. Discard the flowthrough and re-attach the column onto the 2 ml collection tube.

4. Add 500 μl of Buffer NT3 to the column then spin at 11,000 X g for 30 seconds.

5. Repeat step 3, spin the empty column at 11,000 X g for 1 minute to remove residual alcohol in the resin.

6. Remove the 2ml collection tube and attach a new 1.5ml RNAase free microtube. Elute the DNA by adding 40μl of Buffer NE.

   CAUTION: do not touch the resin to eliminate possible cross contamination especially when preparing multiple samples.

7. Incubate the column at room temperature for 10 minutes then spin at 11,000 X g for 1 minute.

   For PCR amplicons greater than 1kb, the column can be incubated at 50°C for 10 minutes

8. Remove the column and measure the DNA concentration by the NanoDrop instrument.

Basic Protocol 8: Deletion of genomic sequence with a pair of guide RNAs

The objective of this basic protocol is to detect the presence of a deletion event following CRISPR editing of DNA sequence between two guide RNA cutting sites. This protocol is appropriate with the assumption that the CRISPR deletion is approximately 500 base pair or less.

1. In order to detect potential deletions two flanking PCR primers should be designed

2. Please see Basic Protocol 5 and Basic Protocol 6 for Primer design, DNA extraction protocol, and gel electrophoresis

3. Each primer should be at least 250 bp away from each potential guide RNA cut site.

4. Please see Figure 8 (second panel) for an example of the various outcomes following CRISPR editing.

Figure 8:

An example of PCR primer design for CRISPR deletion. The sites for sgRNAs for deletion are demonstrated as thunder bolt images showing an estimated 500bp deletion. Any positive sample should carry ~700bp PCR amplification using Pr1 and Pr2 primer set. In the gel picture on the right Sample #1 is showing further deletion (~250bp band) in the other allele in addition of the correct deleted allele (700bp band), whereas #2 shows single amplification of 700bp indicating both alleles carry estimated deletion. #3 sample shows no deletion as it shows 1200bp PCR band comparable to that of control sample (#4). X indicates empty lanes and M indicates DNA ladder (100bp DNA ladder from Promega).

To detect a gene deletion event by CRISPR, we recommend using PCR flanking primers around the target deletion region spanning at least 1kb. This is because there can be greater deletion than the expected. For example, in one case, the expected deletion gap between two guide RNAs (guides 1 and 2) was 240 bp. But the PCR amplicon spanning 1.2 kb around the target region showed that there are some samples with a 500bp deletion, thus the final PCR product size was 700 bp (Figure 8).

When analyzing the image of the agarose gel after PCR reaction for detecting exon deletion, it is critical to compare the PCR products of potential founder mice with that of wild type mice. The positive samples with correct exon deletion will show a PCR band with the size of wild type minus the expected deletion size. A heterozygous positive sample, with one wild type allele and one deleted allele, will show both the control band with deleted band. Biallelic heterozygous positive samples will show only deleted PCR product band. The exact junction for the deletion site for these two alleles might not be always identical as the CRISPR event in each allele is independent of each other. Often there are samples with more than two different PCR bands demonstrating multiple alleles which likely is due to persistent cutting by the ribonucleoprotein complex beyond the two-cell stage. Therefore, it is best to choose founder animals with fewer PCR amplicons.

After the best candidate founder mice are detected by PCR screening the indels can be confirmed by sequencing the PCR products from those animals. To prepare sequencing reactions and align analysis, please refer to Basic Protocol 6.

Basic Protocol 9: Detecting gene knock-in events following CRISPR editing

CRISPR technology has drastically improved the timeline and efficiency to produce gene knock-in animal models by eliminating the lengthy screening, cloning of ES cells, and necessity to breed chimeric mice to detect germline transmission. The types of gene knock-in mice include reporter lines, conditional Cre lines, and humanized animal lines. Depending on the types of gene knock-in, the size of the desired insertion can reach up to several hundred kilobases. Although synthetic oligos and single strand DNA are widely used as repair templates, they are less likely to generate random integration. A plasmid-based replacement template is the better choice for larger insertions. It is therefore critical to differentiate the proper knock-in from the random integration of the repair template and confirm both 5’ and 3’ flanking sequences for the knock-in by initial PCR screening, and subsequent confirmation by DNA sequencing.

Materials

Alcohol Swab (Covidien, cat. no. 6818)

Ring handle ear punch, 5mm (Roboz Surgical Instrument Co., cat. no. 65–9902)

Alcohol resistant marker pen, black (Fisher brand, cat. no. 1400–20-FSC)

DNA lysis buffer (See reagents and solutions):

Tris HCl, pH 8.0 (Thermo Scientific, cat. no. AM9855G)

EDTA (MilliporeSigma, cat. no. E7889–100ML)

Sodium Chloride (MilliporeSigma, cat. no. S5886)

Sodium dodecyl sulfate (MilliporeSigma, cat. no. L3771)

Proteinase K (Thermo Scientific, cat. no. 25530–049)

Isopropyl Alcohol (MilliporeSigma, cat. no. I9516)

Absolute Alcohol (MilliporeSigma, cat. no. 459844

PrimeStar Max polymerase (Clontech, cat. no. R040A)

Nuclease-free water (MilliporeSigma, cat. no. W4502–50ML)

Custom oligonucleotide primers (e.g., eurofinsgenomics.com or idtdna.com; Lyophilized, see reagents and recipe for generating 10 pmol primer)

2% Agarose gel (See reagents and solutions)

Agarose (MP Biomedicals, cat. no. 11AGAH0250)

50x TAE buffer (Thermo Scientific, cat. no. B49

Ethidium Bromide solution (Alfa Aesar, cat. no. J63382)

CAUTION: Ethidium Bromide is a known mutagen. PPE must be used and any downstream waste products must be handled according to institutional guidelines.

NucleoSpin Gel and PCR cleanup (Clontech, cat. no. 740609)

TOPO^™ TA Cloning^™ Kit for Subcloning, with One Shot^™ TOP10 chemically competent E. coli cells (Thermo Scientific, cat. no. K450001)

LB agar plate with 50μg/ml Kanamycin (Quality Biological, cat. no. 340–147-231)

LB broth (Thermo Scientific, cat. no. H2676036)

Kanamycin (Thermo Scientific, cat. no. 450811000)

Rattler Plating Beads (Zymo Research, cat. no. 1154H26):

Smart kit E4 LTS for 20, 100, 1000 μl electronic pipette (Rainin, cat. no. 30386731) or equivalent

Multi-Channel pipette (Rainin, cat. no. pipette Lite XLS 17013803) or equivalent

Mastercycler, nexus gradient (Eppendorf, cat. no. 6331) or equivalent

Refrigerated Centrifuge (Eppendorf, cat. no. 5427R) or equivalent

Vortex mixer (Daigger, cat. no. Vortex-Genie2) or equivalent

AirClean 600 PCR workstation (AirClean Systems, cat. no. AC624) or equivalent

Thermo Scientific^™ Owl^™ A2 Large Gel Systems (Thermo Fisher, cat. no. 09–528-102)

Microwell comb (Thermo Fisher, cat. no. A2-MT2C)

Imaging system (Azure Biosystems, Azure 300)

Microwave (Panasonic, cat. no. S28645) or equivalent

Heat block (Eppendorf, Thermomixer F1.5) or equivalent

Magnetic Stirrer (Fisher Scientific, cat. no. S88857200) or equivalent

Nanodrop 2000 (Thermo Scientific, cat. no. ND2000CLAPTOP)

PCR Tube Strips, 0.2ml (Eppendorf, cat. no. 951010022) or equivalent

PCR plate, semi skirted (Eppendorf, cat. no. 951020346) or equivalent

1.5ml microtubes (Ambion, cat. no. AM12400) or equivalent

Latex or Nitrile gloves (Fisher Brand, cat. no. 11–394-15 or 19–041-171) or equivalent

1000 μl BioClean Ultra tips (Rainin, cat. no. 30389211) or equivalent

200μl BioClean Ultra tips (Rainin, cat. no. 17005859) or equivalent

20μl BioClean Ultra tips (Rainin, cat. no. 17005860) or equivalent

S-Blocks and Round-Well Blocks (Qiagen, cat. no. 19585) or equivalent

AirPore Tape Sheets (Qiagen, cat. no. 19571) or equivalent

Magnetic Bar (Fisher Scientific, cat. no. 14–513-68) or equivalent

500ml flask (Fisher Scientific, cat. no. 41030250) or equivalent

Sample preparation for sequencing analysis to detect knock-in founder mice

We highly recommend that the whole insertion site including flanking sequence should be sequenced in the founder animal to confirm targeted recombination before moving forward with breeding. For this purpose, sequencing is performed on PCR products with a primer that is further 5’ of the homology arm. Otherwise, the PCR product may derive from any randomly integrated flox sequence. In addition, this PCR reaction will result in at least two bands, one from the wild type allele and another from the knock-in allele. Therefore, gel extraction of the desired PCR band is necessary to achieve a good sequencing result. Furthermore, the volume of PCR reactions needs to be increased, since a minimum of 40 ng/μl of PCR product is required for sequencing reactions. See below for an example of preparation for sequencing submission.

PCR amplification

1. Perform the PCR reaction using the Pr2/Pr5 Primer pair (see figure 10).

   It is highly recommended to amplify the PCR product with Pfu polymerase.

2. Scale up the volume of the PCR reaction (between 50 to 100 μl) to have sufficient quantities of amplicon for subsequent sequencing.

Figure 10:

Illustration of primer locations to detect a large insertion by CRISPR, demonstrating different sites of primers. Pr1 and Pr5 are flanking the HDR template and are used in combination with Pr3 and Pr4 respectively, to recognize unique sequence of the insertion to the target site. The combination of Pr2 and Pr3 will amplify an HDR specific band, however this amplicon is within the HDR template DNA, thus not informative for screening for correct region of the genome by HDR template DNA. Note that the 5’HA and 3’HA represents 5’ and 3’ homology arms within the HDR template. The site of DSB by gRNA is shown as a thunder arrow.

Agarose Gel electrophoresis/purifying the expected PCR product

1. Wash the gel apparatus chamber briefly with a 0.1% bleach solution and then place the chamber under running water in a sink for 10 minutes to remove the bleach solution.

2. Wash all the gel apparatus with deionized water for 1 minute then air dry completely.

3. Prepare a 0.6% Agarose gel with a wide comb (See reagents and recipe).

4. Load the entire PCR product in a comb slot along with DNA ladder and apply an electric current at 50 Volts/hour until there is a good separation of the desired band.

Generally, the separation can be observed when the dye in the loading buffer reaches the bottom of the gel.

Gel extraction using the NucleoSpin^® Gel and PCR Clean-up kit.

1. Place the gel on the UV illuminator with a long wavelength setting, quickly slice and remove the desired PCR band using a new razor blade.

   CAUTION: Make sure to wear a UV protective face shield while the UV lamp is on.

2. Place the gel piece in a 1.5 ml microtube.

3. Estimate the weight of the gel piece by subtracting the weight of empty microtube from that of the microtube with the gel piece.

4. Add 2 volumes of NT buffer and dissolve the gel at 60°C for 10 minutes. For example, add 200 μl of NT buffer to a 100mg gel piece. Check the tube periodically to ensure that all the agarose has dissolved and invert the tube couple of times to ensure the gel is completely dissolved into the NT buffer.

5. After NT buffer solution has cooled to room temperature, load the NT buffer and gel mixture onto the provided column and attached the provided 2 ml collection tube to the bottom of the column.

6. Centrifuge the column/collection tube at 11,000Xg for 30 seconds.

7. Discard the flowthrough in the collection tube and add 600 μl of NT3 buffer, centrifuge at 11,000Xg for 1 minute.

8. Discard the flowthrough in the collection tube and place the collection tube back on to the column, centrifuge at 11,000Xg for 1 minute. This is to remove any residual alcohol inside the column.

9. Attach a new 1.5ml RNAase free microtube at the bottom of the column, then add 30 μl of EB buffer to the center of the column.

10. After 10 minutes, centrifuge the column/tube at 11,000Xg for 1 minute.

    It is crucial not to touch inside of the column with the pipette tip to prevent any cross-contamination especially when dealing with multiple samples.

    For amplicons more than 1kb, the column can be incubated at 50°C for 10 minutes

11. Discard the column and measure the concentration using the Nanodrop spectrophotometer.

Basic Protocol 10: Screening of conditional knockout floxed mice

Cre/LoxP-based conditional knockout technology has been a powerful tool for gene function analysis that allows temporal and regional specific gene manipulation. Using CRIPSR to generate flox mice (flanked by LoxP) is widely requested, since approximately 30% of genes are considered essential for development (Bernas, 2012). Yet, inserting a pair of LoxP cassettes remains a challenging task (Gurumurthy, 2019). A two-donor floxing method with a pair of guide RNAs, for example, can lead to a complex set of gene modifications such as single LoxP insertions, double LoxP insertions flipped to a “in trans” configuration, and deletions adjacent to the LoxP site (Fig 12, A, B and C). It is those undesired, compound gene modifications that make the screening for the correctly floxed insertion allele much more difficult than that of any other type of knockin insertion. Therefore, the screening for floxed mice involve numerous exclusion screenings for those un-desired alleles. In the current section, all the screening steps to identify both LoxP insertions in the correct orientation, and located in the correct flanking sequence will be described in detail.

Figure 12:(A,B).

Demonstration of complex set of potential Loxp insertions in different alleles by CRISPR. A. Maximum number of alleles with different gene modifications including wild type (unmodified, bottom bar in black). B. Allele profile of a founder animal with the desired Loxp insertion (in cis) in one of the alleles and one unmodified allele. C. Allele profile of a founder animal with trans-Loxp insertion in each of the two alleles. D. Allele profile of a founder animal with more than two types of alleles due to mosaicism. Note that the desired allele can be established by breeding to wild type animals and screening for mice bearing the genotype in B

Materials

NucleoSpin Gel and PCR cleanup (Clontech, cat. no. 740609)

TOPO^™ TA Cloning^™ Kit for Subcloning, with One Shot^™ TOP10 chemically competent E. coli cells (Invitrogen cat. no. K450001)

LB agar plate with 50μg/ml Kanamycin (Quality Biological, cat. no. 340–147-231)

LB broth (Thermo Scientific, cat. no. H2676036)

Kanamycin (Thermo Scientific cat. no. 450811000)

Rattler Plating Beads (Zymo Research,# 1154H26)

QIAprep Spin Miniprep Kit (Qiagen, cat. no. 27104)

Smart kit E4 LTS for 20, 100, 1000 μl electronic pipette (Rainin, cat. no. 30386731) or equivalent

Multi-Channel pipette (Rainin, cat. no. pipette Lite XLS 17013803) or equivalent

Mastercycler, nexus gradient (Eppendorf, cat. no. 6331) or equivalent

Refrigerated Centrifuge (Eppendorf, cat. no. 5427R) or equivalent

Vortex mixer (Daigger, Vortex-Genie2) or equivalent

AirClean 600 PCR workstation (AirClean Systems, cat. no. AC624) or equivalent

Thermo Scientific^™ Owl^™ A2 Large Gel Systems (ThermoFisher, cat. no. 09–528-102)

Microwell comb (ThermoFisher, cat. no. A2-MT2C)

Imaging system (Azure Biosystems, Azure 300)

Microwave (Panasonic, cat. no. S28645) or equivalent

Heat block (Eppendorf, Thermomixer F1.5) or equivalent

Magnetic Stirrer (Fisher Scientific, cat. no. S88857200) or equivalent

Nanodrop 2000 (Thermo Scientific, cat. no. ND2000CLAPTOP)

PCR Tube Strips, 0.2ml (Eppendorf, cat. no. 951010022) or equivalent) or equivalent

1.5ml microtubes (Ambion, cat. no. AM12400) or equivalent

Latex or Nitrile gloves (Fisher Brand, cat. no, 11–394-15 or 19–041-171) or equivalent

1000 μl BioClean Ultra tips (Rainin, cat. no. 30389211) or equivalent

200μl BioClean Ultra tips (Rainin, cat. no. 17005859) or equivalent) or equivalent

S-Blocks and Round-Well Blocks (Qiagen, cat. no. 19585) or equivalent

AirPore Tape Sheets (Qiagen, cat. no. 19571) or equivalent

Magnetic Bar (Fisher Scientific, cat. no. 14–513-68) or equivalent) or equivalent

Conducting and Analyzing PCR Results to Detect Floxed Founder mice

For a comprehensive screening approach that can identify both flox insertions rather quickly by PCR, we recommend setting up a total of 3 different PCR conditions that can check both sides of the floxed region and to detect evidence of a floxed insertion in cis (Fig. 13, P1/P2, P5/P6 and P3/P4), it is critical that correct positive animals should show the predicted PCR bands in all these 3 PCR sets.

Figure 13(A,B):

Example of screening strategy and PCR result demonstrating wide range of allelic profiles among the offspring. A. Illustration of primer locations to detect two sites of Loxp insertions by CRISPR, demonstrating different locations of primers. P2 and P5 are overlapping 5~6 bp of flanking region, and the LoxP site (noted in the inlet picture, in yellow) and are used in combination with P1 and P6 respectively, to recognize flox insertion into the target site. The combination of P3 and P4 will amplify both flox to flox sequence in the same allele indicating the desired cis-LoxP insertion into the single allele. B. A gel picture displaying the result of this strategy above from 16 potential founder mice. Animals #1 and #14 shows the desired Loxp insertions as all three PCR conditions worked, whereas animals #2, #3, #6, #9 and #10 are inserted with only one Loxp site. The PCR reaction for Animal #4 indicates a trans-loxP insertion. Animal #12 indicates an insertion of the HDR template in a random region.

Preparing Sequencing for Detecting Floxed Founder Mice

Once mice with all 3 PCR positive PCR bands are identified, the sequence of both loxP sites as well as the flanking sequence of the homology arms of the HDR template should be confirmed in the founder animal just as other types of knock-in insertions. The aim of these 3 PCR screens is that there is at least an allele with correct insertion in this founder. It is possible that the founder contains complicated alleles in addition to the correct one (Figure 14B). Therefore, the goal of sequencing the DNA from the founder mouse should be to identify and confirm the correct HDR allele regardless of the complexity. There can be several sequencing strategies depending on the resources available. First, the whole HDR region including both flox sites along with at least one side of flanking sequence to the homology arm can be amplified by PCR using a set of primers then the amplicon can be used as a template for the sequencing reaction (i.e., P1/P6 in Figure 13). However, the result of the sequencing will likely show a difficult to interpret chromatogram due to heterogenous alleles amplified at the same time (Figure 14A). To reduce the complexity in the PCR product, an allele with desired correction can be specifically amplified, then sequencing reactions can be used using flox-specific primers (P1/P4 and P3/P6 in Figure 13). Yet, the region around the flox sequence insertion could still be heterogenous, thus resulting in a non-specific chromatogram. Therefore, the PCR product should be sub-cloned into a plasmid (such as the TOPO cloning vector). Several bacterial colonies can be re-screened following bacterial transformation with flox-specific PCR reactions. The procedure described below is to clone the PCR product into a TOPO cloning vector, bacterial transformation followed by selection of positive clones by the presence of a selectable marker.

Figure 14.

Illustration of TOPO cloning strategy to separate specific alleles from CRISPR founder animals with flox insertion. A. Demonstration of heterozygous chromatograms files of PCR products amplified from CRISPR founder animals. B. Illustration of separation of specific alleles by breeding with wild type animals with founder animals. C. Illustration of separation for specific alleles for sequencing reactions by TOPO cloning

TOPO cloning and bacterial transformation

1. To an RNAase free 1.5ml microtube, add 1μl of salt solution (provided in the kit), 2 μl of PCR product, 2 μl of Nuclease free water (provided in the kit), and 1 μl of TOPO vector (provided in the kit). Incubate the ligation reaction mix at room temperature for 5 minutes.

2. Place competent cells from the TOPO cloning kit for bacterial transformation into an ice bucket for 1 minute then add 2 μl from ligation mix in the prior step. Mix the tube by tapping the side gently.

3. Incubate the competent cells + TOPO cloning mixture at 42°C for exactly 30 seconds (timing is critical) then immediately place the microtube back in the ice bucket.

4. After 5 minutes, add 250 μl of S.O.C. culture media then incubate the tube at 37 °C for 1 hour with shaking at 250 rpm.

5. Spread 50 μl from the above culture onto kanamycin-resistant LB plates using rattler plating beads and incubate at 37 °C overnight.

6. Confirm that there are enough bacterial colonies that are separate from each other such that individual colonies can be picked out without cross-contact from other colonies. If there are too many colonies, then one can re-streak a new LB/kanamycin plate to obtain individual colonies.

Selection of clones

1. For efficient selection of the correct clone with a targeted allele, several colonies should be transferred (with separate pipette tips for each colony) into a PCR plate containing 75 μl of LB broth with 25 μg/ml Kanamycin.

   We recommend increasing the number of inoculated colonies by the size of PCR product (for example, 4 colonies/kb. This is due to the decreasing cloning efficiency relative to the size of the DNA product. For example, if the size of the PCR product is 2kb, inoculate 8 individual colonies to screen.

2. Once inoculated, the PCR plate is incubated at 37 °C with shaking at 250 rpm for 8 hours to overnight.

3. For colony screening PCR, 19 μl of PCR master mix with the primer to detect flox sequence paired with M13 forward or reverse primer is added onto a new PCR plate (For composition, please refer Table 2 from Basic Protocol 9). Using multi-channel pipette, 1 μl of bacterial culture is then added to the master mix, then proceed to the PCR reaction.

   After setting up the PCR reaction, the PCR plate with the bacterial culture should be sealed until the correct clone has been chosen.

4. After gel electrophoresis to determine which clonal DNA has the correct amplified product, prepare 2 ml of LB broth with 25 μg/ml Kanamycin in 6 ml bacterial culture tubes. Carefully open the seal for the PCR plate with the bacterial culture then inoculate 1 μl of chosen clonal culture. The tube will then undergo incubation at 37°C with shaking at 250 rpm for 12 hours to overnight.

Purification of plasmid DNA using the QIAprep Spin Miniprep Kit

1. Aliquot 1.5ml of bacterial culture into a 2ml microtube.

2. Separate the bacterial mass from the culture media by centrifugation at > 7000Xg at room temperature for 5 minutes.

3. Carefully discard the culture media supernatant.

4. Add 250 μl of P1 buffer (provided in the kit) to the tube and resuspend the bacterial pellet until there is an even suspension.

5. Add 250 μl of P2 buffer (provided in the kit) to the tube, then mix by inverting the tube 4–6 times.

   This process should not exceed 5 minutes which will cause shearing of DNA. Optionally, (but recommended), if Lysis blue is added to P2 buffer, the solution turns blue once the lysis of bacterial cells is complete. Invert the tube until you see homogenous blue color throughout the solution.

6. Add 350 μl of N3 buffer (provided in the kit) and mix the tube by inverting the tube 4–6 times.

7. As the lysis solution becomes neutralized, white clumps will appear in the tube. If Lysis blue is added to P2 buffer, then the blue color should disappear completely upon neutralization.

8. Centrifuge the tube at >17,000Xg for 10 minutes. A compact white pellet should form at the bottom of the tube.

9. Carefully transfer the supernatant into the spin column included in the kit then centrifuge at >11,000xg for 30–60 seconds.

10. Discard the flow through, then add 600 μl of PE buffer to wash the column, then spin at >11,000xg for 30 seconds.

11. Repeat the washing process by adding 600 μl of PE buffer then spin for 30 seconds.

12. Centrifuge the empty column for 1 minute to remove the residual alcohol in the column.

13. Attach the spin column to an RNAase free 1.5ml microtube.

14. Add 50 μl of EB buffer provided, to the center of the column to elute the plasmid DNA (being careful not to puncture the column membrane).

15. After incubating 5 minutes at the room temperature, centrifuge the tube at >11,000Xg for 1 minute to collect the DNA to the bottom of the 1.5ml microtube.

16. Measure the concentration of the plasmid by Nanodrop and proceed to DNA sequencing using Sanger sequencing.

Sequencing primers should be designed to cover every 500 to 600 bp including M13 forward and reverse primers which reside in the backbone of TOPO vector, using primer walking (overlapping primers) to generate amplicons downstream of the 600 bp sequence.

REAGENTS AND SOLUTIONS

Blastocyst DNA lysis buffer (stored at room temperature):

50 millimolar (mM) Tris HCl (pH8.0) (1 M Tris-HCl, pH8.0, Teknova, cat. no. T1115 or equivalent)

10 mM KCl (pH 8.3) (Sigma-Millipore, cat. no. P3911 or equivalent)

2.5 mM MgCl₂ (Sigma-Millipore, cat. no. M8266 or equivalent)

0.1 milligram (mg)/ml gelatin

0.45% NP40 (volume/volume)

0.45% Tween-20 (v/v)

1 M Potassium Chloride (KCl, Sigma-Millipore, cat. no. P3911 or equivalent) (Stock)

Dissolve 7.4g of KCl powder to 80 ml of distilled water by stirring with magnetic bar. Once dissolved completely, adjust the final water volume to 100ml. Autoclave the solution at 121°C, 15 lb./sq.in. (psi) for 15 minutes using liquid setting then store at room temperature for up to 2 years.

1 M Magnesium Chloride (MgCl₂, Sigma-Millipore, cat. no. M8266 or equivalent) (Stock)

Dissolve 20.3g of MgCl₂ powder to 80 ml of distilled water by stirring with magnetic bar. Once dissolved completely, adjust the final water volume to 100 ml. Autoclave the solution at 121°C, 15 lb./sq.in. (psi) for 15 minutes using liquid setting then store at room temperature for up to 2 years.

1 mg/ml Gelatin, Sigma-Millipore, cat. no. G2500 or equivalent (Stock)

Dissolve 50mg of Gelatin powder to 50ml of distilled water then filter through 0.22 μg syringe filter, store at room temperature for up to 1 year.

9% Nonidet P-40 (NP-40, Fisher Scientific, cat. no. NC9168253 or equivalent) (Stock)

Add 9ml of NP40 to 91 ml of distilled water into 250 ml beaker, stir until completely dissolved, store at room temperature for up to 1 year.

9% Tween-20, MP Biomedical, cat. no. 11TWEEN201 or equivalent (Stock)

Add 9ml of Tween-20 to 91 ml of distilled water into 250ml beaker, stir until completely dissolved, store at room temperature for up to 1 year.

Recipe for 20 ml of Blastocyst DNA lysis buffer

100μl 1M Tris HCl, pH 8.0

200 μl 1M KCl

200 μl 1M MgCl₂

500 μl 1mg/ml Gelatin

100 μl 9% NP40 (v/v)

100 μl 9% Tween 20 (v/v)

18.75 ml of Distilled Water

This solution can be stored at room temperature for 1 month without Proteinase K 50 μl of Proteinase K (Thermo Scientific, cat. no E00491 or equivalent) should be added before use

Lysis buffer after adding Proteinase K should be used fresh. Discard any remaining solution.

DNA lysis buffer

10 mM Tris-HCl (Invitrogen, cat. no. AM9855G), pH 8.0

100 mM Sodium Chloride, NaCl (Millipore-Sigma, cat. no. S5886)

10 mM EDTA (Millipore-Sigma, cat no. E7889–100ML)

0.5% sodium dodecyl sulfate, Millipore-Sigma, cat. no. L3771)

SDS (weight/volume)

0.2 mg/ml of Proteinase K ,(Thermo Scientific, cat. no E00491 or equivalent)

Reagents for agarose gel electrophoresis

SeaKem LE Agarose (Lonza, cat. no. 50004 or equivalent)

Tris-Acetate-EDTA (TAE), 50X concentration, dilute to 1X with water (Quality Biological, cat. no. 351–008-131 or equivalent)

Ethidium Bromide (EtBr, 10 mg/ml, Invitrogen, cat. no. 15585–011)

USE CAUTION when handling this solution

SYBR Safe, (substitute for Ethidium Bromide),(Invitrogen, cat. no. S33102, 10,000X)

Reagents

1M Tris HCl, pH 8.0 (Invitrogen,cat. no. AM9855G)

0.5M EDTA (MilliporeSigma, cat no. E7889–100ML)

Sodium Chloride, NaCl (MilliporeSigma, cat. no. S5886)

Sodium dodecyl sulfate, SDS (MilliporeSigma, cat. no. L3771)

Proteinase K (Invitrogen, cat. no. 25530–049)

5 M Sodium Chloride (NaCl) (Stock)

Dissolve 29.2 gram (g) of NaCl into 70 ml of distilled water by stirring with magnetic bar. Once the powder is completely dissolved in water, add additional distilled water to a total of 100ml then autoclave at 121°C, 15 lb./sq.in. (psi) for 15 minutes using liquid setting. This stock solution can be stored at room temperature for 1 year.

10% Sodium Dodecyl Sulfate (SDS) (w/v Stock)

Dissolve 10 g of SDS into 100ml of Distilled water until completely dissolved. At times, there may be precipitation of SDS during room temperature storage. Incubate the whole bottle at 37°C until the SDS fully dissolves. This solution can be stored at room temperature for up to 2 years.

Recipe for 10 ml

100μl 1M Tris HCl, pH 8.0

200 μl 0.5M ETDA

200 μl 5M NaCl

500 μl 10% SDS (w/v)

8.9ml Distilled Water

DNA lysis buffer can be stored in room temperature for 1 year without Proteinase K

100μl Proteinase K right before use

After adding Proteinase K, DNA lysis buffer should be used fresh. Discard any unused lysis buffer.

2% Agarose gel

1. Add 6 grams of Agarose to 300 ml of 0.5x TAE (Tris-Acetate-EDTA) buffer in 500ml flask then mix by stirring briefly.

2. Microwave for 1 minute at 1,000 Watts (W) then take out the flask to swirl several times to ensure that the agarose dissolves throughout the TAE. Microwave for 3 more minutes at 1,000W then carefully take out the flask and place the flask onto a stirrer.

3. After the agarose gel mixture is “cool to the touch”, add sufficient Ethidium Bromide to generate a 1% concentration, or SYBR Safe (to a 1X concentration) while the gel mixture is still swirling on the stirrer.

4. Pour the gel mixture slowly in to a sealed lucite gel tray in the gel apparatus.

5. The agarose gel is ready to use after 30 minutes.

The agarose gel can be stored at 4°C for 3 months.

COMMENTARY:

In the first three basic protocol modules, we detail some of the more common methods to purify genomic DNA from mouse tissues. We described a “gold” standard method of tissue digestion followed by phase separation using phenol and chloroform to obtain pure genomic DNA suitable for PCR and even whole genome sequencing. Depending upon institutional guidelines to minimize distress to the mice, there are other methods (e.g., from mouse hair samples) to obtain genomic DNA. This method is a modification of the HotShot protocol (see Basic Protocol 3) where a hot alkaline lysis solution is used to dissolve the hair shaft followed by neutralization, then direct sampling for PCR (Schmitteckert et al., 1999).

Starting in Basic Protocol 5, we describe basic methods to amplify PCR products following different types of CRISPR based editing. We describe how to identify mice that have gene deletions using flanking PCR primers and analysis by gel electrophoresis. In the case of a large deletion (greater than 1 kb), two flanking primers may not be sufficient to span the intervening sequence. In this case, the flanking primer strategy can be modified by designing primers within the un-modified allele (Birling-M-C et al., 2017). The generation of transgenic mice (where foreign DNA is inserted randomly throughout the genome) can present a problem for standard PCR genotyping strategies as the integration site is unknown. As mentioned in basic protocol 5, the integration site can be determined. However, as the transgene can integrate as multiple copies (often in a head-to tail orientation) a method to estimate the copy number is useful particularly when deciding which founder line to maintain. An estimation of copy number can be made using quantitative real-time PCR. In this case, genomic DNA from a “wild-type” mouse has known amounts of the transgene added to generate a standard curve. By comparing cycle thresholds of the unknown founder mice to the cycle thresholds of the known samples, an estimation of the copy number can be made (Shepard, C.T.et al., 2009). For copy number estimations which do not require high sensitivity, SYBR green fluorophores are sufficient. In the case where higher sensitivity is necessary, a probe such as Taqman (Thermo Fisher Scientific) may be necessary. The Taqman probe should also be coupled with a probe with a defined copy number and should not “cross react” to the transgene in question.

Critical Parameters and Troubleshooting

Genomic DNA purification from mouse blastocysts following CRISPR editing

The developmental stage for mouse embryos in one culture plate can vary amongst all the collected embryos. It is critical to collect fully mature blastocysts to obtain a suitable amount of DNA for PCR. As the total volume of the DNA extraction from a blastocyst is 10 μl, adding more then 1ul drop of M2 media along with a blastocyst at the time of collection can significantly dilute the composition of extraction buffer. This will result inefficient extraction of DNA from the blastocyst thus failure of PCR reaction.

T7E1/Surveyor assays to detect indels (insertion or deletions) following CRISPR editing

Each PCR should be optimized with blastocysts from a wild-type mouse prior to using this assay on CRISPR-edited blastocysts to make sure there will be one specific PCR band. It is equally important to amplify a robust PCR band for clear image analysis.

Detection of off-target mutations

The best PCR amplicon size of to detect small indels is around 300–500 bp for further DNA sequencing. To narrow down a potentially large number of off-target genes, a short list can be generated by excluding regions that are not crucial for gene expression, or if a single nucleotide polymorphism (SNP) is present amongst the offspring.

Deletion of mouse genomic DNA deletions with a pair of guide RNAs

Each PCR should be tested with a DNA sample from wild type mouse before the screening to ensure that there will be one specific PCR band. It is crucial to include both positive and negative control samples for PCR amplification.

Detecting gene knock-in events following CRISPR editing

The sequencing analysis should be performed using a PCR product containing flanking regions of homology arms. This is to eliminate any amplified PCR band from randomly integrated HDR template DNAs. The PCR band from the edited allele is significantly diminished compared to the of wild type allele. We highly recommend sub-cloning the PCR band into a PCR based cloning vector (such as a T/A cloning vector) from the edited allele if the band is significantly faint.

Screening of conditional knockout floxed mice

As the loxp sequence is a palindrome, designing specific primers for PCR is critical for the successful screening of a floxed allele. Depending on the complexity of DNA sequence flanking the flox insertion, it is necessary to vigorously test PCR conditions using the HDR template as positive control and wild type DNA as negative control before performing the PCR screen. The sequencing analysis should be performed using the PCR product containing both flanking regions of homology arms. This is to eliminate any amplified PCR band from randomly integrated HDR template DNAs.

Primer design

We recommend testing the efficiency of the genotyping PCR before generation of founder mice. While primer design software tools have made it easier to create a PCR based method for genotyping, the selected primers often still need to be tested to ensure proper amplification of the expected amplicon. To start, usually it is best to select 3 forward, and 3 reverse primer pairs

Troubleshooting variables in genotyping

Troubleshooting purification of the double stranded DNA template

Very low purified template DNA (phenol/chloroform purification, Basic Protocol 1):

One frequent mistake is the use of isopropanol in Basic Protocol 1 instead of isopentyl alcohol. This can result in poor yield or quality of purified genomic DNA . The final absorbance ratio of 260 nm (nanometer) for nucleic acid, to 280 nm for protein should be 1.9. A ratio of 2 or above indicates the presence of RNA. RNA contamination may impede proper PCR amplification. Following analysis using the spectrophotometer, there should be a high amplitude peak at 260 nm with no or very small peaks at 280 or 230 nanometers. A second cause of poor yield is improper tissue size. If the tail biopsy or ear punch biopsy is small, then one solution is to reduce the volume of 1X Tris EDTA in the final rehydration step. An additional tip to ensure genomic DNA purity is to ensure that there is a clear separation between the aqueous phase (containing genomic DNA) and the higher density organic phase where lipids and carbohydrates are dissolved in chloroform. Avoid sampling the interphase emulsion (thin white layer) which may contaminate the genomic DNA. In the event of a large pellet in step 18 of Basic Protocol 1, increase the volume of 1XTE to ensure that there is an even distribution of genomic DNA.

Troubleshooting potential problems (PCR reactions) see table 9

Table 9.

Troubleshooting problems following PCR amplification (Basic Protocols 5 through 10)

Protocol	Problem	Possible Cause	Solution
Basic Protocol 5	No amplicon generated following PCR	Failing to add one or more component of the PCR reaction mix	Add each component sequentially with the Taq polymerase added last
		Degraded genomic DNA	Using gel electrophoresis, analyze a small portion. A smear indicates degraded DNA. A high molecular weight “blob” indicates intact genomic DNA
		Too much genomic DNA template	Dilute the genomic DNA Approximately 20 ng/ μl- 100 ng/ μl is ideal
		Annealing temperature is too high preventing binding of primers to the template	Lower the annealing temperature by 1°C or 2°C and repeat the PCR reaction
		Too few amplicons generated	Increase the cycle number
		Generation of incomplete amplicon	Increase the extension time
		One or more components of the master mix is missing or degraded	Use primers to amplify a gene present in all mouse tissue. For example, amplify the mouse beta globin gene. If no amplicon is detected then modify the PCR reagents or genomic DNA purification method
	Generation of additional non-specific amplicons	Annealing temperature is too low	Verify the optimal annealing temperature and raise the annealing temperature by 1°C or 2°C. Alternatively use a gradient PCR cycler to determine the optimal annealing temperature
		Too high concentration of primers	Reduce the concentration of primers per reaction
		Too high concentration of magnesium chloride	Use a PCR buffer solution with no magnesium chloride and then supplement with additional magnesium chloride until the desired amplicon is detected
		Too many cycles	Reduce the cycle number
		False amplicons in all samples	Use a “no template” control containing water instead of genomic DNA. An amplicon should not be detected in this sample
		Sequence of genomic DNA to be amplified has a high number of G and C bases or repeats of G and C	Additives such as betaine or dimethyl sulfoxide (DMSO) can improve specificity
Protocol	Problem	Possible cause	Solution
Basic Protocol 4	Failure to amplify PCR band	Diluted blastocyst extraction buffer during embryo collection	Minimize the volume of M2 media when collecting blastocysts
		Incomplete extraction of DNA from blastocysts	Make sure to vortex the tube before and after the extraction
			Make sure the blastocysts are submerged in the extraction buffer
Basic Protocol 6	Failure to detect cleavage in the positive control	Inefficient T7 endonuclease	Make sure the enzyme is not expired and try a new vial
	Detection of cleavage in the negative control	Non-specific PCR amplification	Use wild-type genomic DNA to test PCR conditions to amplify a specific PCR product
Basic Protocol 7	Too many off-target genes	The candidate guide RNA’s have a lower specificity score	Select the same chromosome or linked chromosomal region (within 20 Mb) in the in silico analysis
Basic Protocol 5 and Basic Protocol 8	Failure to detect the appropriate PCR band in a sample	Bad quality or insufficient DNA purification	Try to amplify a housekeeping gene (see beta-globin PCR parameters)
		Gene editing created a deletion away from the primer binding sites	Design primers further away and repeat the PCR reaction
Basic Protocol 9	Inconclusive sequencing results due to multiple chromatogram peaks in each amplicon	A complex gene edit occurred in the mouse genome	Perform TOPO cloning and sequence more than one colony
	Failure to amplify both 5’ and 3’ sides of the insertion	There is and indel mutation at one or more of the primer binding sites	Design primers further away and repeat the PCR reactions
	Multiple bands are detected in the DNA sample	The sequencing result is complex	Perform TOPO cloning and sequence more than one colony
Basic Protocol 9 and Basic Protocol 10	Detection of a PCR band in the negative control	One or more of the primer binding sites is not specific or too long	Re-design new primers and repeat the PCR reactions
	Inconclusive sequencing results dure to multiple chromatogram peaks in the sample	A complex gene edit occurred in the mouse genome	Perform TOPO cloning and sequence more than one colony
	Multiple bands are detected in each sample	A complex gene edit occurred in the mouse genome	Perform TOPO cloning and sequence more than one colony

Understanding results:

Analyzing PCR results to detect knock-in founder mice

Please refer to Basic Protocol 1 for the method to collect samples, DNA extraction and Basic Protocols 5-7 for PCR reactions. Since there is no positive control sample for PCR amplification by the knock-in detection primer sets (primer pairs Pr1/Pr3 and Pr4/Pr5 in Figure 10) it is hard to reach a conclusion when no amplification was observed after the PCR. This could indicate there is truly no positive CRISPR animal, or simply the PCR condition is not optimized. In this case, an internal primer pair can be used to amplify to confirm the integration of HDR template (Pr2/Pr3 in Figure 11) along with the positive control sample (the HDR template itself). Although a positive band from internal primer pair could indicate random integration at distinct chromosomes, we recommend to further analyze the animals with positive band from this PCR reaction with an internal primer pair. Additionally, a primer set with both primers located on the outside of HDR template region can be also used to detect the whole insertion (Pr1/Pr5 in Figure 10). In this case however, due to the large size of insertion compared to that of wild type, the PCR product for inserted allele will be substantially fainter. Finally, when analyzing PCR result, the size of PCR band should be carefully compared to the DNA ladder. Any deviation in the estimated PCR band size could imply additional indels during the CRISPR event.

Figure 11(A,B).

An example of sequencing analysis showing locations of primers for primer walking and chromatogram analysis. A. Snapshot from multiple sequence alignments compared against HDR template sequence inserted in a precise genomic location in the mouse genome. Note that the Pr1 and Pr8 primers are designed to sequence the junction of the HDR template with flanking genome sites. A total of 8 sequencing reactions designed to detect the insertion size of 2.5 kb. B. Snapshot from multiple sequence alignments compared against the HDR template sequence showing detailed chromatograms from different sequencing files over the same region the genome of one founder animal. (The blue line indicated in Fig 11A is the start of the region covered by Fig 11B).

For typical labs, sequencing reactions will be done by an outside source (private companies or core facilities in the institute). Once the purified PCR product has been obtained, send the samples to the appropriate outside source following the facilities’ guidelines. We advise setting up both forward and reverse sequencing reactions for one region analysis in case one of them fails to demonstrate a reliable chromatogram. An example for the location of the primers for sequencing is shown in Figure 7.

Figure 7 (A,B):

Schematic diagram for sequencing reactions for off-target regions. Either Pr3 or Pr4 can be used as sequencing primers based upon the PCR amplicon from Pr1 and Pr2. Screenshot of SnapGene program demonstrating a sequencing result by aligning original sequence (top) with Sample 1 and 2. The sequence difference between original sequence and sample2 is shown as red highlighted box.

After receiving files from sequencing reaction output, one should analyze the sequence by aligning the result files with control files. There are several programs to align DNA sequences (Table 6). As an example, the process of DNA alignment with multiple samples to a reference file is shown using the SnapGene program (Figure 8). For each off-target gene, a reference genome sequence and the sequencing result file will be aligned. If there are any differences between these two files, the chromatogram for the sequencing result file should be examined as a confirmation.

Primer set up for sequencing analysis

Typically, the peaks of chromatogram from the first ~100 bp and last ~200 bp of one sequencing reaction are not reliable, and the average size of one sequencing reaction result is about 800 bases. Therefore, it is recommended to set up sequencing reactions for every 500~600 bp. This is so called “primer walking”, and the purpose is to cover the regions across both 5’and 3’ flanking sequence (Figure 11).

To analyze the proper insertion of the cassette with the flanking sequence, all the sequencing files from the primer walking sequencing should be aligned together against one control file which is generated at the time of design the construct. Please refer Basic Protocol 8 for the information regarding software programs for multiple alignment. An optimal display of multiple alignments is shown in Figure 11. Since the first ~200bp and the last ~200 bp of sequence chromatogram can be ambiguous, it is important to set up enough overlaps among each reading to clarify the correct sequence.

Analyzing Sequencing Results to detect gene knock-in events

Once the sequencing results are received, all the files (text and chromatogram) should be aligned to a reference sequence which contain the sequence of both 5’ and 3’ flanking as well as HDR template. As an example, the process of DNA alignment with multiple samples to a reference file is shown using SnapGene as described in Basic Protocol 9. For the sequencing reactions using PCR product from the founder animal, check the chromatogram to see if there is a second ranked peak that matches with reference file. If there are any discrepancies between the result and the reference file, this may indicate a modification. This homoduplex chromatogram is observed in the samples with heterozygosity. If the sequencing reactions are done with clones using plasmid preparation, there should be no homoduplex chromatogram.

Screening of floxed alleles

The following are several examples to interpret the PCR results. If the sample only shows positive bands with internal flox specific PCRs, that indicates random integration of HDR templates (Fig. 13B, sample #12). If the flox sequence was inserted in trans, all the other PCRs would work but there will be no PCR band with the pair of 5’ flox specific forward and 3’ flox specific reverse primer (Fig. 13, sample#4). The sample might show positive PCR bands except the condition with one side of flox specific primer paired with HDR flanking primer. This means that there is an allele with only one side of flox correctly and there is also a random integration of HDR template in the same animal.

Time Considerations for DNA purification and PCR based genotyping (please see Table 10)

Table 10:

Time considerations for DNA purification and PCR-based genotyping (Basic Protocols 1 through 10)

Protocol	Process	Step	Approximate elapsed time
Basic Protocol 1	Purification of mouse genomic DNA using phenol/chloroform	Digestion of tissue biopsy	Minimum of 4 hours For ear punch biopsies or preferably overnight (12 hours) with a large biopsy
	Purification of mouse genomic DNA using phenol/chloroform	Phenol/chloroform extraction	2 hours (for approximately 18 samples)
Basic Protocol 2	Purification of mouse genomic DNA using the Maxwell 16 instrument	Biopsy sample to purified genomic DNA	45 minutes
		Quantitation of concentration in a spectrophotometer	20 minutes

Protocol	Process	Step	Approximate elapsed time
Basic Protocol 3	Isolation of crude genomic DNA using the HotShot method	Alkaline lysis, heating, and neutralization	1 hour
		Quantitation of concentration in a spectrophotometer	20 minutes
Protocol	Process	Step	Approximate elapsed time
Basic Protocol 4	Genomic DNA isolation from mouse blastocysts following CRISPR edits	Collection of blastocysts	2 minutes per blastocyst
	Genomic DNA isolation from mouse blastocysts following CRISPR edits	DNA extraction from blastocysts	45 minutes
Basic Protocol 5 and Basic Protocol 6	T7E1/Surveyor assays to detect indels following CRISPR editing	T7E1 nuclease treatment	2 hours
Basic Protocol 7	Detection of off-target mutations	Sequencing (Sanger)	6 hours “in house” 2 days to outsource to a commercial entity
		PCR product sub-cloning	2 days
Basic Protocol 8	Deletion of genomic DNA sequence with a pair of guide RNAs	PCR	90 minutes
		Gel electrophoresis	1 hour
Basic Protocol 9	Gene knockin using CRISPR	PCR	90 minutes
		Gel electrophoresis	1 hour
Basic Protocol 10	Screening of conditional knockout (floxed) mice	PCR	90 minutes
		Gel electrophoresis	1 hour

Strategic Planning to genotype various GEM:

Indels, Point Mutations, and Protein Tags:

In this case, only flanking primers to a mutation are typically needed, so different combinations of the primers can be tried using only wild type mouse tail DNA. Select the primer pair that generates the clearest PCR band for further genotyping.

Large Inserts via Homologous Recombination:

Unlike the above smaller genetically engineered mutations, designing primers to genotype large gene insertions is more complicated. Primers that can flank the whole expanse of the genetic insert are often not favorable as these PCR reactions may need span thousands of base pairs. Thereby, to confirm proper recombination, a PCR is generally designed with one primer is located 5’ outside the homology arm of the donor DNA and the other primer within the inserted sequence (i.e., a drug resistance selection marker or a knocked in GFP). The homology arms needed to promote homologous recombination typically begin around at least 0.5 kb, but can be much longer, so a long PCR product still needs be generated just to detect proper targeting. To test the efficacy of the primers, one may want to have a positive control plasmid that is diluted in wild type mouse genomic DNA (only 0.1–1 ng of plasmid DNA is often needed for PCR). The positive control plasmid may, for example, include extended sequence beyond the homology arm and part of the insert. Upon confirmation of the proper recombination, a shorter more favorable PCR amplicon of 200–1000 bp can be designed for routine genotyping purposes, still with the one primer located within the inserted sequence. A PCR program to detect the homozygous mutant, heterozygous mutant, and wild type can later be designed as well. Usually, in this case, a set of 3 primers are used in this PCR reaction – a single forward primer, for example, with one reverse primer for the wild type allele and another reverse used for the targeted inserted mutation. With Primer-BLAST, a designated shared primer can be set in primer parameters so that the software only finds a corresponding opposite primer. This 3 primer PCR design may require a lot of optimization. Start with PCR for one allele, select the primers that work best, and then, with the newly selected shared primer, test the parameters needed to amplify the other allele. Lastly, combine all 3 primers in one reaction, where primer concentrations may need to be adjusted to get PCR bands from both alleles of equal intensity on an agarose gel.

Transgenic Mice:

A genotyping PCR is designed with primers inside the transgene, and a test to examine the efficacy of the primers can be done with the transgenic plasmid (about 0.1–1 ng)

Designing Primers for the T7E1/Surveyor nuclease assay:

There is a list of primer design tools including free web versions. (In this section, Primer-BLAST (see internet resources)). For clear detection of PCR bands in the gel imaging step, about 800–1,000 bp amplicon is recommended for this assay. The following are several key points when designing primers for this assay.

Designing primers to detect point mutations and other small mutations

To generate a point mutation, a homologous recombination template such as oligonucleotide (ODN) with the desired mutation along with a mutated PAM site (to prevent recutting) is used along with guide RNA. To detect the correct insertion of the HDR template by PCR, the location of one of the primers should be in the flanking site to the homology arm of the HDR template and another primer will be a mismatch primer where the 3’ side of primer sequence is in the mutation site (Figure 9). A silent mutation to create a novel restriction enzyme site can be designed for easier diagnostic screening.

Figure 9:

Primer design scheme to detect point mutations by CRISPR. Note that the Pr1 is upstream of the HDR template and Pr2 is designed to be specifically recognized by the mutation (enlarged box detail of sequence composition for mismatch PCR primer, Pr2).

Larger HDR templates

For larger insertions, such as a reporter or Cre recombinase cassette, longer HDR templates, such as plasmid or long single stranded DNA (ssDNA) with the desired insertion sequence along with much longer homology arms, are used with guide RNA for CRISPR editing. To confirm the insertion of the HDR template in the correct region of the genome, flanking sequence to HDR specific region should be amplified by PCR reactions using one primer located in the flanking sequence of HDR insertion and another primer that specifically recognizes the HDR sequence (i.e., GFP or Cre recombinase) in both of 5’ and 3’ side of the insertion (Pr1/Pr3 and Pr4/Pr5 in Figure 10). It is highly recommended to check both 5’ and 3 ‘site of insertions by PCR to ensure proper insertion.

Design Primers to Detect floxed Knockin by PCR

There have been several different strategies to generate flox insertions depending on the size of the gap between two LoxPs, availability for technical resources, and the expected timeline. One of the strategies is to introduce two individual oligonucleotides each containing a flox site along with corresponding guide RNAs. This reagent mix can be either microinjected or electroporated into fertilized embryos (Miyasaka et al., 2018). This “CLICK” method however can result in large genomic deletions between the flox gap while still inserting the correct flox sequence. Therefore, to circumvent the deletion, long ssDNAs, which contain both flox sites and CRISPR ribonucleoproteins, are either microinjected or electroporated into fertilized embryos (Miura et al., 2017). This Easi-CRISPR method however is only suitable for flox insertion gaps of less than 1.5kb due to a limitation in the of size of generating this type of ssDNA (Hirofumi et al., 2017). Another way to prevent the deletion, is to insert oligonucleotides with each flox site along with corresponding guide RNAs consecutively (Bernas et al., 2022). Finally, a HDR template plasmid or lssDNA that contain both flox sites as well as longer homology arms can be microinjected with a single guide RNA (Gurumurthy et al., 2019).

Regardless of the strategy, the main goal of PCR screening is to detect if two short oligonucleotides or a single HDR template that were used for the insertion of both LoxP sites were inserted in the correct region. To design primer pairs to detect flox insertions in the correct regions, one of the primers should reside on the LoxP site and the flanking sequence. The placement of the flox specific primer is crucial for detecting both floxed regions. A total of 4 different flox specific primers can be designed to detect all sides of LoxP sequence at both 5’ and 3’ ends (Fig. 13, P2, P3, P4 and P5). When designing flox specific primers, the 3’ region of flox specific primer should consist of 4–5 bp of flanking sequence followed then by 22bp of LoxP sequence (Fig. 13, inlet pic). The other primer in this pair should be placed flanking sequence of HDR template (Fig. 13). The PCR screening using this kind of primer pair should be done for LoxP sites. Finally, a PCR reaction using both flox specific primers is performed to detect flox insertion in cis in the same allele (Fig. 13, P3/P4). As a side note, these flox specific primers can be tested for its effectiveness by detecting the correct PCR band amplified from HDR template such as plasmid donor when paired with primer that reside within the plasmid (Fig. 13, P0/P4).

Table 1:

Advantages and Disadvantages of various genomic DNA purification methods

Method	Cost	Advantage	Disadvantage
Phenol/Chloroform extraction	$	Exceptional purity Can be used for multiple applications	Time consuming for multiple samples, corrosive agents
Proteinase K/isopropanol	$	Quicker than phenol/chloroform	Crude purification may not be optimal for sensitive applications
Maxwell 16	$$$$	Almost fully automated, quick, high purity	Initial high cost to purchase the system, must use cartridges, quantity of purified DNA is limited for applications requiring large amount of template (e.g. whole genome sequencing)
HotSHOT	$	Can be used with a variety of samples including semen, buccal swabs	Crude purification suitable for PCR only, corrosive agents

Table 8.

List of online sequence analyzing tools

Software	Cost	Multiple alignment	Link
SnapGene	Paid	v	https://www.snapgene.com/snapgene-viewer
Geneious	Paid	v	https://www.geneious.com/
SeqMan Pro	Paid	v	https://www.dnastar.com/software/lasergene/molecular-biology/
Sequencher	Paid	v	http://www.genecodes.com/
VectorNTI	Paid	v	https://www.thermofisher.com/us/en/home/life-science/cloning/vector-nti-software.html
Chromas Pro	Paid	v	http://technelysium.com.au/wp/chromaspro/
CodonCode Aligner	Paid	v	https://www.codoncode.com/aligner/index.htm
DNA Baser	Paid	v	http://www.dnabaser.com/
GeneStudio Pro	Free	v	http://genestudio.com/
UGENE	Free	v	http://ugene.net/
Chromaseq	Free	v	https://mesquiteproject.org/packages/chromaseq/manual/
BioLign	Free	v	http://en.bio-soft.net/dna/BioLign.html
4Peaks	Free		https://nucleobytes.com/4peaks/index.html
Sequence Scanner	Free		http://resource.thermofisher.com/page/WE28396_2/
Chromas Lite	Free		http://technelysium.com.au/wp/chromas/

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.