CRISPR/Cas9-Mediated Introduction of Specific Heterozygous Mutations in Human Induced Pluripotent Stem Cells

Abstract

Advances in genome editing and our ability to derive and differentiate human induced pluripotent stem cells (hiPSCs) into a wide variety of cell types present in the body is revolutionizing how we model human diseases in vitro. Central to this has been the development of the CRISPR/Cas9 system as an inexpensive and highly efficient tool for introducing or correcting disease-associated mutations. However, the ease with which CRISPR/Cas9 enables genetic modification is a double-edged sword, with the challenge now being to introduce changes precisely to just one allele without disrupting the other.

In this chapter, we describe strategies to introduce specific mutations into hiPSCs without enrichment steps. Monoallelic modification is contingent on the target activity of the guide RNA, delivery method of the CRISPR/Cas9 components and design of the oligonucleotide(s) transfected. As well as addressing these aspects, we detail high throughput culturing, freezing and screening methods to identify clonal hiPSCs with the desired nucleotide change. This set of protocols offers an efficient and ultimately time- and labor-saving approach for generating isogenic pairs of hiPSCs to detect subtle phenotypic differences caused by the disease variant.

1. Introduction

Over the last two decades, few technologies have impacted the stem cell field more profoundly than the development of techniques to generate induced pluripotent stem cells (iPSCs) and to perform targeted genome editing using clustered regularly interspaced short palindromic repeat (CRISPR)-based systems [1–3]. The ability to derive human (h)iPSCs from specific patients has enabled a wide repertoire of diseases to be studied in vitro, specifically in the cell types affected in the patient [4, 5]. While these models are providing crucial insights into the pathogenesis of rare genetic diseases, confounding factors arising from the derivation of the hiPSC lines as well as differences in genetic background can impair the ability to detect a disease phenotype.

These issues can be addressed by either correcting the known mutation in the patient-derived hiPSCs or by introducing the genetic variant into a wild-type (control) hiPSC line; thereby generating isogenic pairs of cell lines that in principle differ only by the putative disease-causing mutation. Such modifications have been greatly facilitated by the development of endonuclease-based gene editing systems, with the CRISPR/Cas9 system gaining traction due to the ease with which the system can be adapted to readily target different genomic sites.

The CRISPR/Cas9 system consists of a nuclease (Cas9) and generally a synthetic guide RNA (sgRNA) composed of a variable 20 nucleotide sequence (crRNA) which determines DNA-binding specificity, fused to a transactivating RNA (tracrRNA) that mediates the association of Cas9 nuclease with the crRNA/tracrRNA complex [2]. Once transfected into hiPSCs, the Cas9 nuclease is guided by the sgRNA to the complementary DNA sequence in the genome and when this sequence is followed by a 3′ protospacer adjacent motif (PAM), Cas9 will create a blunt-ended double-strand break (DSB). Cas9 orthologs from different species require different PAM sequences, the most commonly used Cas9 being isolated from the bacterium Streptococcus pyogenes with a PAM sequence of 5′ -NGG-3′. The resulting DSB activates the DNA repair machinery to correct the lesion either via the error-prone non-homologous end joining (NHEJ) pathway which can result in insertion or deletion mutations (indels), or via homology-directed repair if a template with sequences homologous to the region around the DSB is provided [6]. To generate precise sequence modifications, a DNA construct such as a single-stranded oligonucleotide (ssODN) can be used as the template for homology-directed repair [7].

While procedures to introduce knockout mutations in hiPSCs via the dominant NHEJ repair pathway are relatively robust, precise and scarless monoallelic targeting remains a challenge. This is in part due to the prerequisite that the DSB needs to occur at a minimal distance (ideally <20 bp) from the desired modification [8], thereby impacting the selection of possible sgRNAs that meet these requirements. Aside from the sgRNA needing to have low predicted off-target activity, the on-target efficiency of the sgRNA is also critical. Very efficient sgRNAs can make it difficult to identify clones in which one allele contains the desired modification and the non-targeted allele is without nonspecific indels. Likewise, sgRNAs with low on-target activity can make it problematic to identify modified clones unless an enrichment step is included. Differing approaches based on the activity of the sgRNA are therefore required to improve the likelihood of obtaining monoallelic targeted clones without disrupting the other allele or leaving residual exogenous sequences within the locus.

Here we describe the strategies we have used to introduce specific heterozygous missense mutations into a well-characterized wild-type hiPSC line and the resulting protocols used to identify and isolate clonal lines differing only by the introduced variant [9]. Importantly, and prior to undertaking the targeting experiment, various assays and screens should be performed and established to streamline and minimize the labor-intensive aspects of the procedure (Fig. 1). These include: (1) the development of a robust DNA screening strategy for evaluating potentially several hundred clones concomitantly; (2) assessing in vitro the on-target activity of the sgRNAs generated, and based on this; (3) the method to deliver the CRISPR/Cas9 reagents as well as the ssODN to the hiPSCs.

Fig. 1. Workflow outlining assays and screens to establish prior to performing the genetic modification of the hiPSC line.

(a) This includes establishing the gDNA isolation method and PCR conditions for amplifying the region surrounding the target site. The sequencing information is then used to design sgRNAs no further than 30 bp from the target site and synthesize them by in vitro transcription. (b) The on-target activity of the resulting sgRNAs is then evaluated in the target hiPSC line by TIDE analysis of the sequencing traces obtained following PCR amplification of the target region in the electroporated hiPSCs. Based on this information ssODNs are designed to introduce the intended mutation, as well as silent mutations for screening or preventing disruption of the second allele. The relevant procedures in Subheading 3 for each step are indicated

We have found that sgRNAs determined by TIDE analysis [10] to have an on-target activity of >40% efficiency, show a very high percentage of biallelic targeting when delivered by electroporation. However, liposome-based transfection methods or the electroporation of a second ssODN that contains silent mutations to prevent the sgRNA recognizing and cutting it, can result in monoallelic targeted cells being relatively easily isolated. For sgRNAs with on-target efficiencies estimated to be between 10% and 40%, electroporation of the sgRNA and Cas9 nuclease along with the ssODN containing the desired base alteration will likely result in a mixture of clones that are either bi- or monoallelic targeted at a frequency that no more than 100 colonies should need to be screened. If the sgRNA appears to be less than 10% efficient, this could indicate that the chromatin structure surrounding the genomic region is not permissive for introducing DSBs and/or cutting leads to gene lethality. Testing further sgRNAs, adjusting the targeting strategy to include a selection step, or using a different Cas9 ortholog or designer nuclease should be considered as alternative strategies if targeting that specific site in the gene is required.

The steps following transfection are lengthy and can lead to the potential screening of several hundred clones. We also explain how by altering additional nucleotides in the ssODN, restriction fragment length polymorphism (RFLP) analysis can be part of the screening procedure and can be performed directly after transfection. This serves as a checkpoint to decide whether to proceed with subcloning or repeat using a different transfection strategy. We provide protocols that enable 200 or more subclones to be easily processed both for DNA isolation and cryopreservation by a single operator, and also afford the researcher with the time required to comprehensively screen and validate any putative positive clones. Finally, we describe how to thaw the resulting monoallelic targeted hiPSC clones (Fig. 2).

Fig. 2. Workflow for transfecting, screening and recovering hiPSCs with targeted heterozygous mutations.

This includes the transfection strategy to deliver the Cas9-sgRNA RNP and ssODN(s) to the hiPSCs, followed by evaluating via RFLP screening whether to proceed with clonal isolation of the transfected hiPSCs. If this is the case, the resulting colonies are replicated with part of the clone cryopreserved and the remainder used for further characterization of the genetic modifications that have occurred within the target region. Clones identified as having the desired modifications are then recovered and expanded from the cryopreserved cells. The relevant procedures in Subheading 3 for each step are indicated

2. Materials

2.1. General Molecular Biology Reagents

1. Proofreading DNA polymerase (e.g., Q5® High-Fidelity, Platinum™ Taq High-Fidelity or PrimeSTAR DNA Polymerase).

2. dNTPs.

3. RNase-free H₂O.

4. DNase I, RNase-free.

5. Cas9 protein (10 μg/μl, IDT).

6. TE buffer: 10 mM Tris pH 8.0, 0.1 mM EDTA.

7. DNA Extraction Solution (e.g., QuickExtract™, Lucigen).

8. Exonuclease I.

9. Shrimp Alkaline Phosphatase.

10. Thermocycler.

11. 0.2-ml PCR tubes.

12. 96-well PCR plate.

13. Equipment and reagents for performing agarose gel electrophoresis.

2.2. sgRNA In Vitro Transcription (IVT)

1. DNA template of tracrRNA sequence (e.g., Addgene #62988 vector).

2. DNA purification kit (e.g., QIAquick PCR Purification Kit, Qiagen).

3. DNA extraction kit.

4. HiScribe™ T7 High Yield RNA Synthesis Kit (New England Biolabs).

5. RNA purification kit (e.g., NucleoSpin RNA Clean-up XS kit, Macherey-Nagel).

2.3. hiPSC Culture

1. 100 × RevitaCell™ Supplement (Gibco).

2. Culture medium: StemFlex™ Medium (Gibco), Penicillin– Streptomycin (50 units/ml).

3. Dilution medium: DMEM/F12 + HEPES, 0.5%BSA (Bovine serum albumin).

4. Phosphate buffered saline without CaCl₂ and MgCl₂ (PBS^−/−).

5. Human recombinant laminin-521 (LN521).

6. Accutase cell detachment solution.

7. 1 × TrypLE Select Enzyme.

8. Primocin antimicrobial reagent (50 mg/ml).

9. FACS wash solution: 2% (v/v) fetal bovineserum(FBS), 2mM EDTA in PBS^−/−.

10. 4′,6-Diamidino-2-Phenylindole (DAPI; Invitrogen): 0.1 mg/ ml in FACS wash solution.

11. 5 ml test tubes with 35 μm cell strainer.

12. KSR: KnockOut™ Serum Replacement (Gibco).

13. Dimethyl sulfoxide (DMSO).

14. Isopropanol.

15. Freezing medium: KSR with 20% (v/v) DMSO.

16. Mineral oil sterile (e.g., #M5310, Sigma-Aldrich).

17. 6-, 24-, 48-, and 96-well cell culture plates.

18. 0.75 ml Matrix™ Alphanumeric storage tubes (ThermoFisher).

19. Freezing container for storage tubes.

2.4. Transfection Reagents

1. 0.5 ml low protein binding tubes (Eppendorf).

2. Neon™ Transfection System with 10 μl Neon electroporation tips (ThermoFisher).

3. Resuspension buffer R (ThermoFisher).

4. Opti-MEM™ I Reduced Serum Medium (Opti-MEM; ThermoFisher).

5. Lipofectamine™ Stem Transfection Reagent (Invitrogen).

3. Methods

3.1. PCR Screening Strategy and sgRNA Design

1. Seed the hiPSC line to be modified in 4–5 wells of a 96-well plate at densities between 3.2 × 10⁴ and 1.6 × 10⁵/cm² (see Note 1).

2. When cells are at least 25% confluent, aspirate the medium and wash the wells with 200 μl of PBS ^−/−.

3. Add 30 μl of QuickExtract™ DNA Extraction Solution to each well. Pipette the solution up and down to lyse the cells and then transfer the contents to a PCR tube. Add 30 μl of QuickExtract solution directly to one PCR tube to serve as a no template control (NTC) (see Note 2).

4. Extract the genomic DNA (gDNA) by placing the PCR tubes in a thermocycler and running the following program:

   65 °C for 15 min;

   68 °C for 15 min;

   98 °C for 10 min.

5. Store the samples at −20 °C.

6. Design 2 or 3 pairs of forward and reverse primers that bind at least 500 bp 5′ and 3′ from the target sequence to be modified (see Note 3) (Fig. 3).

7. Test the primer pairs on 1–2 μl of the isolated gDNA using a PCR program based on the protocol recommended by the manufacturer of the DNA polymerase and appropriate for the size of the amplicon to amplified.

8. Run 3–5 μl of each PCR product on an agarose gel to confirm the expected amplicon is obtained.

9. Purify the PCR product by transferring 5 μl of each PCR product to a PCR tube and adding 2 μl Exonuclease I and 2 μl Shrimp Alkaline Phosphatase (see Note 4).

10. Place the PCR tubes in a thermocycler and incubate using the following parameters:

    37 °C for 30 min;

    80 °C for 30 min.

11. Perform Sanger sequencing on the treated PCR product (see Note 5).

12. From the Sanger sequencing results, copy a sequence of approximately 60 bp (30 bp 5′ and 3′ from the intend modified region) and paste as input into the online guide RNA design tool CRISPOR (http://crispor.tefor.net/) [11]. Select the genome GRCh38, and the PAM 20 bp-NGG−Sp cas9. Submit the job to identify potential guide sequences.

13. Select at least four guide sequences based on the specificity score and predicted off-targets (see Note 6).

Fig. 3. Schematic illustrating the design of the sgRNAs and primers within the target locus.

Guide sequences of the sgRNAs are selected based on their specificity scores and predicted off-targets using online design tools, and are a maximum of 30 bp (5′ or 3′) from the nucleotide(s) to modify (red mark). Screening primers (gray arrows) are designed to generate an ~1,000 bp amplicon with the target site near the middle of this sequence. Nested sequencing primers (green arrows) are designed within the amplicon and are ~200 bp from the target region (light blue box) to improve the likelihood of having a sequence read of good quality around the cut site of the sgRNA

3.2. sgRNA Generation by IVT

1. For each guide sequence selected, order an oligonucleotide comprising of the following sequence:

   5′-tgt aat acg act cac tat ag-(N)18-20-gtt tta gag cta gaa ata gc −3′.

   where the sequence at the 5′ end (bold letters) corresponds to the T7 promoter, the sequence at the 3′ end corresponds to part of the tracrRNA, and (N)_18-20 is the 18–20 bp guide sequence (see Note 7).

2. In addition, order an oligonucleotide with the following sequence (see Note 8):

   5′-agc acc gac tcg gtg cca ct −3′.

3. Reconstitute the oligonucleotides in TE buffer to a stock solution of 100 μM.

4. Prepare a 10 μM working solution of the oligonucleotides by further diluting in ddH₂O an aliquot of the stock solutions. To generate a DNA template of each sgRNA, set up a PCR (total volume 50 μl) comprising of the following:

   15 ng pSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid (Addgene #62988).

   200 μM dNTPs.

   0.2 μM of each primer.

   1 × enzyme buffer.

   Nuclease-free H₂O to 49.5 μl.

   0.5 μl Q5 Polymerase.

5. Perform the PCRamplification using the following conditions:

   98 °C−30 s; [98 °C−10 s; 72 °C−20 s] × 30 cycles; 72 °C− 2 min.

6. Run 1–2 μl of the PCRproduct on a 2% agarose gel to confirm a product of ~110 bp.

7. Purify the remaining PCR product using a DNA purification kit. Elute in RNase-free H₂O(see Note 9).

8. Determine the concentration of the purified PCR product using a spectrophotometer. If required, adjust with additional RNase-free H₂O so that the final concentration is between 30 and 90 ng/ μ l.

9. Set up an IVT reaction (20 μl) for each sgRNA. Assemble the reaction in the following order (see Note 10):

   1.5 μl 10X reaction Buffer.

   1.5 μl each NTPs (7.5 mM each NTP final). Template DNA (up to 1 μg).

   1.5 μl T7 RNA polymerase mix. Nuclease-free H₂Oto20μl.

10. Incubate in a thermocycler at 37 °C for 16 h.

11. Degrade the DNA template by treating the IVT reaction with 2 μl DNase I in 78 μl nuclease-free H₂O (total volume 100 μl). Incubate in a thermocycler at 37 °C for 15 min (see Note 11).

12. If the procedure needs to be paused, freeze the reaction at −20 °C. Otherwise proceed with purifying the sgRNAs using an RNA purification kit (see Note 12).

13. To determine the concentration of the sgRNAs accurately, make a dilution series (1:10; 1:20; 1:40; 1:80) of each sgRNA and analyze using a spectrophotometer. Take the average of the measurements that give similar concentrations when the dilution factor is considered. If necessary, dilute the stock sgRNA using RNase-free H₂O so that the final concentration is between 500 and1,000 ng/μl. Reconfirm the concentration of the stock sgRNA solution.

14. To check the quality of the 110 bp sgRNA, run 100 ng on a denaturing TBE-Urea PAGE gel as described elsewhere [12].

15. Make 10 μl aliquots of each sgRNA and store at −80 °C until required.

3.3. Determining the On-Target Activity of the sgRNAs

1. To test how efficiently each sgRNA cuts the gDNA target sequence, electroporate the sgRNAs along with Cas9 protein into the hiPSC line to be modified.

2. 24–48 h prior to electroporation, passage the hiPSCs such that cells are 70–90% confluent on the day of the experiment (see Note 13) (Fig. 4).

3. Based on the number of sgRNAs to be tested, determine the total number of electroporations to be performed. Include also an electroporation of Cas9 without a sgRNA as the control sample for sequencing. Calculate the quantity of hiPSCs and amount of Cas9 protein and sgRNA that will be required. Each electroporation requires 1.5 × 10⁵ hiPSCs, 1.5 μg Cas9, and 360 ng sgRNA (see Note 14).

4. Prepare a 48-well plate for replating the electroporated hiPSCs. Coat the number of wells required with LN521 at a surface density of 0.5 μg/cm² and incubate the plate at 37 °C for 2 h. Aspirate the LN521 solution and add 250 μl of culture medium (without antibiotics) containing RevitaCell™ Supplement (1:100). Incubate the plate in a 37 °C, 5% CO₂ cell culture incubator.

5. Pre-warm to room temperature (~18–20 °C): PBS^−/−; Resuspension Buffer R and Electrolytic Buffer (Buffer E) from the Neon transfection kit.

6. PrepareaCas9-sgRNARNPcomplexforeachsgRNA.

7. In 0.5 ml low protein binding tubes, dilute the total amount of Cas9 protein required to 1 μg/μl in Resuspension Buffer R(see Note 15).

8. Dilute each of the sgRNAs to 240 ng/μl in Resuspension Buffer R.

9. In a 0.5 ml low protein binding tube add, in the following order:

   1.5 μl of 1μg/μl Cas9.

   1.5 μl of 240 ng/μl sgRNA.

   4.5 μl Resuspension Buffer R.

10. Incubate the Cas9-sgRNA RNP complex at room temperature for a minimum of 10 min but no longer than 1 h.

11. During the incubation, prepare the hiPSCs and Neon transfection system for electroporation.

12. Wash the hiPSCs with PBS^−/− and harvest the cells using 1x TrypLE Select. Dilute the cell suspension with 4 × the volume of Dilution medium and take an aliquot to determine the cell concentration.

13. Pellet the hiPSCs by centrifugation (300 × g, 3 min) and resuspend in PBS^−/−. Transfer the total number of cells required to a 1.5 ml microcentrifuge tube and pellet by centrifugation (300 × g, 3 min). Resuspend the cells in Resuspension Buffer Rat a final density of 2 × 10⁷ cells/ml; gently pipetting to obtain a single cell suspension (see Note 16).

14. To each Cas9-sgRNA RNP complex add 7.5 μl of the cell suspension. Mix by pipetting up and down gently.

15. Connect the Neon® Pipette Station to the Neon® device and fill the Neon® Tube with 3 ml of Buffer E (see Note 17).

16. Load the electroporation tip into the Neon pipette based on the manufacturer’s instructions. Gently pipette the cell-RNP complex mixture twice before taking up 10 μl(see Note 18).

17. Electroporate the cells using default electroporation protocol #8 (1300 V, 30 ms, 1 pulse) (see Note 19).

18. Immediately transfer the electroporated cells into a well on the 48-well plate containing the prewarmed medium. Gently rock the plate to evenly distribute the cells. Repeat steps 16 and 17 for each cell-RNP complex. Place the plate in a humidified 37 °C, 5% CO₂ cell culture incubator.

19. Check whether the hiPSCs have attached after 24 h. If most cells have attached, refresh the culture medium and return the plate to the incubator; otherwise leave for a further 24 h before refreshing the culture medium.

20. When the hiPSCs are at least 50% confluent, harvest the cells as described in step 12 (see Note 20) (Fig. 4).

21. Resuspend the cell pellet in 200 μl PBS^−/− for gDNA isolation. The cells can be processed either immediately or stored at −20 °C.

22. Transfer 100 μl of each cell suspension to a 0.2 ml PCR tube. Centrifuge at 300 × g for 5 min at room temperature to pellet the cells.

23. Aspirate the PBS^−/− and add 30 μl of QuickExtract DNA extraction solution. Pipette the solution up and down to lyse the cells (see Note 21).

24. Amplify the edited genomic region using the PCR conditions established in Subheading 3.1 (steps 4–8).

25. Purify the PCRproduct using a PCR purification kit.

26. Perform Sanger sequencing on the treated PCR product. To estimate the on-target cutting efficiency of each sgRNA, upload the sequence trace files for both the control sample as well as the sgRNA-transfected sample to the TIDE website (https://tide.nki.nl/) and analyze as previously described [10]. Repeat for each sgRNA transfected (see Note 22).

27. Determine which sgRNAs have the highest on-target activity and, based on the efficiency, decide the optimal strategy for achieving precise monoallelic modification of the locus (Table 1).

Fig. 4. Phase contrast images showing the appropriate confluency of hiPSCs to be harvested for electroporation (left image), and the recovery of the cells over time following electroporation.

The replated hiPSCs are ~25% confluent 24 h after electroporation (middle image) and ~50% confluent at 72 h (right image). The elongated morphology of the hiPSCs seen at 24 h is due to the presence of RevitaCell™ Supplement in the culture medium. Once removed the cells form more compact and rounded colonies, as depicted at 72 h. Scale bar: 250 μm

Table 1. Strategies to introduce specific heterozygous missense mutations in hiPSC according to sgRNA efficiency.

TIDE-estimated sgRNA efficiency	<10%	10-40%	>40%
Strategy	Design new sgRNAs	Electroporation with mutationcontaining ssODN	Lipofection with mutationcontaining ssODN
	Design a targeting construct that includes a positive selection marker		Electroporation with both a mutation-containing and wild-type ssODN
	Try alternative Cas9 orthologs

3.4. ssODN Design

1. Design the ssODN for introducing the point mutation via homologous recombination based on the most efficient sgRNA determined in Subheading 3.3.

2. Homology arms should include 40–80 bp of sequence adjacent to the sgRNA cut site, with the point mutation to introduce being at least 30 bp from the ends of the ssODN. The total length of the ssODN should be between 120 and 150 bp (Fig. 5).

3. To prevent alleles that have undergone homologous recombination with the ssODN from being re-cut by the Cas9, introduce a silent mutation within the “GG” part of the PAM sequence. If this is not possible, introduce 1 or 2 silent mutations in the sequence corresponding to the seed region (8–10 nucleotides proximal to the PAM) of the sgRNA.

4. Incorporate additional silent mutations within the ssODN to create a novel restriction endonuclease recognition site to assist in identifying potential targeted clones during the screening procedure by RFLP analysis. Use the web tool WatCut (http://watcut.uwaterloo.ca/template.php) to identify potential silent mutations to create such a restriction site within 15 nucleotides either side of the sgRNA cut site (see Note 23).

5. If the estimated efficiency of the sgRNA is >40% and the nucleotide to be modified is within 20 bp of the Cas9-induced DSB, a second ssODN that does not contain the point mutation should also be designed (wild-type ssODN). This ssODN should contain the same silent mutations to disrupt the binding of sgRNAas the modifying ssODN. Ifpossible, try to incorporate a different novel restriction endonuclease recognition site to that in the mutation-containing ssODN for RFLP screening analysis (see Note 24).

6. Order the ssODNs with standard desalting purification. Resuspend in TE buffer at a stock concentration of 100 μM. Store at −20 °C.

Fig. 5. Schematic illustrating the design of the ssODNs for use as templates for homology-directed repair.

The total length of ssODNs are ~120–150 nucleotides (nt), with homology arms being between 40-80 nt relative to the Cas9 cut site (indicated by break in the target locus). If possible, introduce a silent mutation within the “GG” part of the PAM sequence to avoid modified alleles being re-cut by Cas9. Also include additional silent mutations within 15 nt of the sgRNA cut site to create restriction enzyme sites for use in RFLP screening. Finally, the ssODN should contain the mutation of interest to introduce and, if required to prevent disruption of the other allele, a second ssODN corresponding to the wild-type sequence should also be designed ideally with different silent mutations for RFLP screening

3.5. Targeted Introduction of Specific Mutations in hiPSCs−-Electroporation Procedure

1. For a 10 μl transfection that is subsequently plated in a 48-well cell culture plate, we electroporate (per well) 1 × 10⁵ hiPSCs, 1 μg Cas9 protein, 240 ng sgRNA, and 40 pmol ssODN(s) (see Note 25).

2. Prepare the hiPSCs and reagents required for performing the electroporation and replating cells as described in steps 2–5 in Subheading 3.3.

3. Dilute the ssODN(s) to 13.3 μM in Resuspension Buffer R.

4. Dilute the Cas9 protein and sgRNA as described in steps 7 and 8 in Subheading 3.3.

5. In a 0.5 ml low protein binding tube add in the following order:

   1.5 μl of 1μg/μl Cas9.

   1.5 μl of 240 ng/μl sgRNA.

6. Incubate the Cas9-sgRNA RNP complex at room temperature for a minimum of 10 min but no longer than 1 h.

7. During the incubation of the RNP complex, harvest the hiPSCs for electroporation as described in steps 12 and 13 in Subheading 3.3.

8. Add 4.5 μl of diluted ssODN to the RNP complex and mix by pipetting up and down.

9. Immediately add 7.5 μl of cell suspension to the above mixture.

10. Proceed with the electroporation and plating as described in steps 15–19 in Subheading 3.3.

11. When the cells are 70% confluent (Fig. 6), harvest the hiPSCs using 1 × TrypLE Select and replate a fraction to maintain the transfected lines for subcloning (Subheading 3.8). Keep the remaining cells (~5–8 × 10⁴ cells) for gDNA isolation to assess the bulk population for targeting (Subheading 3.7).

Fig. 6. Phase contrast images showing differences in the recovery of the replated hiPSCs depending on the electroporation conditions and components transfected.

Images with a black border indicate the timepoint at which the transfected cells are sufficiently confluent (>70%) for passaging. Cells electroporated with the Cas9-sgRNA RNP complex and ssODN using protocol #8 were not yet sufficiently confluent for passaging at 72 h. Additionally hiPSCs electroporated using these conditions as well as with protocol #6 would not have a medium change for the first 48 h due to the lower cell recovery. Scale bar: 250 μm

3.6. Targeted Introduction of Specific Mutations in hiPSCs−Lipofection Procedure

1. 24–48 h prior to transfection, seed the hiPSCs in a 24-well cell culture plate such that cells are ~60% confluent on the day of the experiment (see Note 26) (Fig. 7).

2. On the day of transfection, change the medium on the hiPSCs to 500 μl Opti-MEM plus RevitaCell™ Supplement (1:100) per well just prior to preparing the reagents. Return the cells to the 37 °C, 5% CO₂ cell culture incubator.

3. Dilute the Cas9 protein and sgRNA in Opti-MEM to the final concentrations of 1 μg/μl and 380 ng/μl, respectively.

4. Form the Cas9-sgRNA RNP complex by combining 1 μl sgRNA with 1.5 μl Cas9 protein in a 0.5 ml low protein binding tube. Incubate at room temperature for at least 10 min but no longer than 40 min prior to adding to the cells. After10 min add 22.5 μl Opti-MEM (final volume 25 μl).

5. Dilute the ssODN to 4 pmol in 25 μl Opti-MEM (160 nM).

6. In a microcentrifuge tube, add 57.5 μl of Opti-MEM medium followed by 5 μl of Lipofectamine™ Stem Transfection Reagent directly into the medium. Mix by flicking and incubate at room temperature between 5 and 20 min (see Note 27).

7. Divide the diluted liposomal mixture across two 0.5 ml tubes (25 μl each).

8. Add 25 μl of the RNP complex into one of these tubes and 25 μl of the 160 nM ssODN into the other tube. Mix the solutions by flicking the tubes.

9. Incubate both mixtures at room temperature for 5 min.

10. To each well containing hiPSCs, add dropwise 50 μl of the liposome-RNP complex, followed by dropwise 50 μl of the ssODN lipoplex solution. Swirl the medium in the wells while adding the mixtures to ensure distribution over the entire surface. Place the plate in a humidified 37 °C, 5% CO₂ incubator.

11. After 4 h, supplement each well with 500 μl ofculture medium. Refresh with new culture medium 24 h later.

12. When the cells are 80–90% confluent, harvest the hiPSCs, and replate a fraction to maintain the lines for subcloning (Subheading 3.8) (Fig. 7). Keep the remaining cells (~1–1.5 × 10⁵ cells) for gDNA isolation to assess the bulk population for targeting (Subheading 3.7).

Fig. 7. Phase contrast images showing the appropriate confluency of the hiPSCs prior to lipofection with the Cas9-sgRNA RNP complex and ssODN (left image), and confluency at which to harvest the transfected cells for passaging (right image), which is typically 48 h after transfection. Scale bar: 250 μm.

3.7. Screening the Bulk Population of Transfected hiPSCs

1. Isolate gDNA using a gDNA extraction kit from the leftover transfected cells (Subheadings 3.5 or 3.6).

2. Amplify the edited genomic region using the PCR conditions established in step 7 of Subheading 3.1.

3. Using the RFLP screening procedure established to indicate targeting, digest 2–3 μl of the PCRproduct with the appropriate restriction enzymes and run on an agarose gel to indicate whether targeting has occurred.

4. Proceed to subcloning if the above screen indicates that targeted hiPSCs are present in the transfected population of cells (see Note 28).

3.8. Clonal Isolation of Transfected hiPSCs by Single Cell Deposition

1. Seed two wells of a 12-well plate with the transfected hiPSCs from Subheadings 3.5 or 3.6 such that the cells will reach ~70% confluency 2 days later (see Note 29).

2. 3 h before single cell deposition, coat 4x 96-well plates with 1.8 μg/cm² LN521 using a repeat pipettor. Incubate the plates at 37 °C for at least 2 h (see Note 30).

3. Meanwhile, prepare 40 ml of culture medium containing RevitaCell™ Supplement (1:100) and Primocin (200 ng/ml) (see Note 31).

4. Aspirate the LN521 solution from a 96-well plate and immediately replace with 100 μl/well of the culture medium prepared in step 3 using a repeat pipettor. Repeat for each of the plates (see Note 32). Store the plates in 37 °C, 5% CO₂ cell culture incubator until required.

5. Harvest the transfected hiPSCs using 1x TrypLE Select. Combine the cells from the 2 wells together. Pellet the cells by centrifugation (300 × g, 3 min) and resuspend in 1 ml of FACS wash solution.

6. Filter the cell suspension using a 5 ml tube with a 35 μm cell strainer cap. Rinse the filter with an additional 1 ml of FACS wash solution.

7. Cover the cell strainer cap with Parafilm and centrifuge the tube for 1 min at 480 × g to pellet the hiPSCs.

8. Carefully aspirate the supernatant and resuspend the cells in 550 μl of FACS wash solution. Transfer 500 μl of the cell suspension to a sterile FACS tube and add sterile DAPI to a final concentration of 0.1 mg/ml. Add 150 μl FACS wash solution to the remaining 50 μl cell suspension to use as an unstained gating control.

9. Using a flow cytometer with sorting capability, deposit a single viable (DAPI⁻) cell into each well of the 96-well plates prepared in step 4. Place the plates in a humidified 37 °C, 5% CO₂ cell culture incubator, and do not disturb for 72 h (see Note 33).

10. 3 days after single cell deposition, replace the medium in each well with 150 μl of culture medium containing 200 ng/ml Primocin using a repeat pipettor. Repeat this refreshment every 3 days.

11. Cell outgrowth should be visible under a microscope 7–10 days after subcloning. Mark the wells that contain colonies (see Note 34).

12. Replicate the colonies (Subheading 3.9) 12–14 days after single cell deposition.

3.9. Replicating Clonal hiPSCs Cultured in 96-Well Plates

1. Identify the wells containing colonies that will be passaged (see Note 35).

2. For each colony identified, coat two wells on two separate 96-well plates with LN521 at a surface density of 1.5 μg/ cm². Incubate the plates in a 37 °C, 5% CO₂ cell culture incubator for 2 h. Label one of the plates “Archive” and the other plate “DNA” (see Note 36).

3. Remove the LN521 solution from the wells and replace with 100 μl culture medium. Place the plates in a 37 °C, 5% CO₂ cell culture incubator until required.

4. Take one of the four 96-well plates with hiPSC clones to replicate. Aspirate the medium from the marked wells and wash the cells with 200 μl PBS^−/− (see Note 37).

5. Remove the PBS^−/− and add 30 μl of Accutase to each well. Place the plate in a cell culture incubator for a maximum of 10 min. After 8 min, inspect the wells to see whether most cells are detached. Gently tap the side of the plate to aid in dislodging the hiPSCs (see Note 38).

6. Add 200 μl of culture medium containing RevitaCell™ Supplement (1:100) to each well.

7. Starting with the first clone to be passaged and working quickly, pipette 100 μl of the medium up and down to generate a single cell suspension. Avoid creating air bubbles.

8. Using a microscope, confirm that the clone has been dissociated into single cells.

9. Transfer 100 μl of the cell suspension to a well on the “Archive” plate, and the remainder to the corresponding well on the “DNA” plate. Gently rock the plate to evenly distribute the cells (see Note 39).

10. Repeat steps 7–9 to process the remaining dissociated clones.

11. Repeat steps 4–10 to process the remaining 96-well plates.

12. Place both the “DNA” and “Archive” plates in a 37 °C, 5% CO₂ cell culture incubator.

13. The next day, refresh all the wells with 100 μl of culture medium.

14. Repeat the medium change every second day until the cells are processed either for screening (Subheading 3.10) or cryopreservation (Subheading 3.11).

3.10. Screening the hiPSC Clones

1. Aspirate the medium from the “DNA” plate. Wash the cells with 200 μl of PBS^−/− using a multichannel pipettor.

2. Add30 μl of DNA extraction solution to each well. Usinga multichannel pipettor, pipette the solution up and down ~3 times to lyse the cells. Transfer the contents to the corresponding wells on a 96-well PCR plate.

3. Add the DNA extraction solution to one of empty wells inthe PCRplatetoserveasaNTC.

4. Seal the plate with adhesive foil and vortex briefly (~15 s).

5. Incubate the samples in a thermocycler to extract the gDNA using the parameters established in step 4 of Subheading 3.1.

6. Store the plate at −20 °C until ready to perform the PCR.

7. Set up the PCRin a 96-well PCRplate based on the amplification conditions established in Subheading 3.1; step 7. Use a multichannel pipettor to transfer 1–2 μl of gDNA as template for each reaction.

8. Using a multichannel pipette load 5 μl PCR product on an agarose gel to confirm amplification of the targeted region (see Note 40).

9. Set up the RFLP assay(s) established to identify targeted clones. Make digest reaction solutions (22 μl per clone) comprising of the number of units of restriction enzyme required to cut 1 μg DNA, 1x of the relevant enzyme digest buffer and nuclease-free H₂O. Using a repeat pipettor, transfer 20 μlof the solution into each well of a 96-well PCR plate.

10. Use a multichannel pipettor to transfer 2 μl of each PCR product to the corresponding wells in the digestion plate. Mix by pipetting up and down 2–3 times. Pulse centrifuge the plate.

11. Incubate the plate in a thermocycler following the restriction enzyme requirements recommended for optimal digestion.

12. Run the digests on an agarose gel to identify putative targeted clones.

13. For any clones that appear to be targeted, purify and sequence the remaining PCR product as described in steps 9–11 in Subheading 3.1. Analyze the resulting sequence traces to confirm heterozygous targeting (see Note 41).

3.11. Cryopreservation of Clonal hiPSCs from 96-Well Plates

1. Prior to processing the subclones, prepare enough aliquots of all reagents required.

2. Prepare a container for freezing the 96-well rack holding the storage tubes. Place a layer of paper towels on the bottom of the receptacle and add sufficient isopropanol to saturate these and have some liquid visible. Place the container at 4 °C to cool.

3. Per clone, 150 μl of 2x freezing medium is required. Prepare enough Freezing medium for n+10 clones and pipette 150 μl into each of the storage tubes using a repeat pipettor. Keep the rack containing the storage tubes on ice.

4. Aspirate the medium from the “Archive” plate and wash the cells with 200 μl of PBS^−/−.

5. Using a repeat pipettor, add 30 μl of Accutase to each well and place in the cell culture incubator for 8 min.

6. Add 150 μl of KSR to each Accutase-treated well. Using a multichannel pipettor, pipette up and down three times to generate a single cell suspension.

7. Transfer the cell suspension to the storage tubes containing the 2x Freezing medium. Mix by carefully pipetting up and down 3–4 times (see Note 42).

8. Use a multichannel pipettor to overlay each sample with 125 μl of Mineral oil.

9. Place the 96-well rack containing the storage tubes in the freezing container and then into a −80 °C freezer. Transfer the 96-well rack to liquid nitrogen the next day for long term storage (see Note 43).

3.12. Thawing

Targeted Clonal hiPSCs

1. Prior to thawing the subclones, prepare enough aliquots of all reagents required.

2. For each targeted hiPSC clone to thaw, coat a well on a 48-well plate with LN521 at a surface density of 1 μg/cm² for 2 h at 37 °C.

3. Retrieve the storage tubes containing the clones of interest from liquid nitrogen storage. Thaw at room temperature under sterile conditions.

4. Remove the overlay of mineral oil and transfer the cell suspension to a 15-ml tube (see Note 44).

5. Slowly add dropwise 1 ml of Dilution medium while gently flicking the tube. Add a further 4 ml of Dilution medium dropwise using a serological pipet.

6. Centrifuge the cells at 300 × g for 3 min and resuspend the cell pellet in 500 μl of culture medium containing RevitaCell™ Supplement (1:100) by pipetting the cells up and down three times.

7. Aspirate the LN521 solution and transfer the cell suspension into a well on the earlier prepared 48-well plate. Gently rock the plate to evenly distribute the cells. Repeat for any additional thawed clones. Place the plate in a 37 °C, 5% CO₂ cell culture incubator.

8. The next day, refresh the cells with 500 μl of culture medium per well.

9. When the hiPSCs are >80% confluent, passage and expand the clones.

   10. Isolate gDNA from the passaged cells to reconfirm the introduced modification. Also confirm that the line is negative for mycoplasma. Finally make a frozen stock of the cell line and perform additional characterization of clones (see Note 45).

4. Notes

1. The screening of clones following transfection and single cell deposition is performed in 96-well plates. Therefore, we advise to also use this format when establishing the screening strategy.

2. We have found no gDNA isolation protocol that provides suitable template DNAfor all PCRscreening strategies. Therefore, different gDNA isolation methods as well as different DNA polymerases might need to be tested. There are various solutions for directly isolating gDNA from cells including homemade solutions such as a chloride-based lysis solution [13] or HotSHOT buffer [14], as well as commercial reagents such as QuickExtract™ or DirectPCR lysis solution. We have had most success using QuickExtract™ DNA Extraction Solution to isolate the gDNA and performing the PCR amplification with either PrimeStar (Takara Bio) or Platinum Taq HiFi (ThermoFisher) polymerases. If the expected amplicon is not obtained using these reagents, first try to optimize the reaction by adjusting the PCR conditions or use a different DNA polymerase (e.g., Q5, NEB or TerraDirect, Clontech). If there is still no success, try the other listed procedures for isolating the gDNA.

3. The primers are typically 20–23 nucleotides in length with an ~50% G+C content. The web tool Primer3Plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi) can be used to design the pairs.

4. Exonuclease I removes unincorporated primers, while Shrimp Alkaline Phosphatase dephosphorylates the remaining dNTPs. Although the treated PCR product will still contain some contaminants, this generally does not affect the Sanger sequencing reaction. This enzymatic method is also preferable to columnbased purification methods for later when potentially large numbers of clones may need to be sequenced as part of the screening procedure.

5. Prior to designing in silico the sgRNAs and ssODNs for targeting, itis necessary to knowwhether the sequence of the cell line matches the human reference genome. This can be confirmed by Sanger sequencing the amplicons from the screening PCRs. Primers used for sequencing are internal (“nested”) to the screening pair of primers to avoid sequencing any nonspecific amplicons present in the PCR product and should be at least 200–250 bp from the target sequence to modify. Often one of the other pairs of primers initially designed for screening can be adopted for sequencing. If the cell line is found to be heterozygous for a SNP within the amplicon, it is recommended to try and incorporate this SNP in the final screening and sequencing strategy of the targeted clones as it can assist in determining that no large deletions have occurred in the non-targeted allele.

6. While there are many online tools available for identifying guide sequences, we prefer CRISPOR because it ranks the hits based on in silico specificity and provides information regarding the predicted efficiency, off-targets and known SNPs. However, we have found the predicted efficiency scores oftendo not match the observed on-target activity when evaluated in vitro. Therefore, we do not consider this parameter when selecting guide sequences.

7. When designing the oligonucleotide containing the guide sequence consider that T7 RNA polymerase requires two guanines (G) at the end of the promoter sequence to facilitate efficient transcription. If the guide sequence does not begin with a guanine, an additional one should be added before to the 5′ end of the guide sequence. Truncated guides of 18 or 19 nucleotides also can be designed. In this case additional guanines can be added to make the guide 20 nucleotides in length.

8. This oligonucleotide is a common reverse primer that will anneal to the tracrRNA sequence in the plasmid template. We suggest using Addgene plasmid #62988 as the template, but any vector containing the tracrRNA component is suitable.

9. We use the QIAquick PCR Purification Kit and always add 10 μl of 3 M sodium acetate, pH 5.0, to ensure the optimal pH for binding of DNA to the silica membrane. DNA is eluted in 30 μl of RNase-free H₂O, which is left on the silica membrane for 1 min before centrifugation.

10. The NTPs are sensitive to freeze–thawing. Aliquot each of the NTPs(~5 μl per aliquot) when first thawed and re-freeze. After thawing and using an aliquot, discard anything remaining. A final concentration of 0.75 × of reaction buffer is used when working with short transcripts (<0.3 kb).

11. The EnGen sgRNA Synthesis Kit, S. pyogenes (NEB) can be used as an alternative method to generate the sgRNAs. This is a quicker and simpler protocol to that described here, but the yield following purification is lower (~10–30 μg vs. 25–50 μg).

12. We purify the sgRNAs using the NucleoSpin RNA Clean-up XS kit according to manufacturer’s instructions. Elute the sgRNA in 10 μl of RNase-free H₂O.

13. For maintenance of the hiPSC lines, cell culture plates are coated with LN521 at a surface density of 0.5 μg/cm² based on the manufacturer’s protocol and the cells cultured in StemFlex medium containing penicillin–streptomycin. For passaging, the hiPSCs are dissociated with 1x TrypLE Select Enzyme and collected in Dilution medium.

14. This is calculated based on 1.5 × the volume required per electroporation to ensure there is sufficient. The leftover should be approximately 5 μl/electroporation.

15. In the electroporation mixture the total volume of solutions that are not Resuspension Buffer R should be less than 10% of the total transfection volume (i.e., 1 μl for a 10 μl electroporation). Therefore, the stock solution of Cas9 protein should be >10 μg/μl so that it can be diluted in Resuspension Buffer R.

16. Avoid storing the cell suspension for more than 15–30 min at room temperature as this will reduce cell viability and transfection efficiency.

17. The Neon® Tube containing Buffer E can be used for up to 10 electroporations but should be replaced ifdifferent cell lines are being electroporated to avoid cross-contamination.

18. Visually inspect the tip for any bubbles that were introduced during the loading process. If bubbles appear, re-aspirate the mixture into the tip ensuring there are NO bubbles. Air bubbles can cause arcing during electroporation and lead to an inefficient or failed transfection.

19. We have found this protocol to be the most efficient for electroporating the Cas9-sgRNA RNP complex into hiPSCs, however this might vary between cell lines. For better cell recovery we recommend electroporation protocol #6 (1100 V, 30 ms, 1 pulse) or #13 (1100 V, 20 ms, 2 pulses). The appearance of small air bubbles at the bottom of the tip as well as a “popping” noise indicates that the sample was electroporated. Do not use the same Neon® Tip more than twice because the repeated application of electric pulses reduces the tip quality and impairs their physical integrity. Replace the electroporation tip for different cell lines or sgRNAs to prevent cross-contamination.

20. Usually the cells will be 50% confluent with 36–48 h, and 100% confluent ~72 h after electroporation. It is not necessary to perform a cell count at this step.

21. Use the gDNA extraction solution previously determined to be compatible with the PCR screening conditions established in Subheading 3.1.

22. Ifthe quality of resulting sequence trace files is too poor to be analyzed by TIDE, better quality gDNA can be obtained in step 23 by using a gDNA column extraction kit.

23. Ifpossible, incorporate silent mutations that generate a restriction enzyme site that is otherwise absent from the ssODN but present elsewhere in the PCR screening amplicon. This then confirms that the corresponding restriction enzyme can cut the unpurified PCR product. If such a design is not possible, choose silent mutations that will generate a recognition sequence for a frequently used restriction enzyme. The NEB website provides an overview of the activity of their restriction enzymes in various PCR buffers (https://www.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-pcr-buffers). However we recommend still to confirm the activity of the restriction enzyme in the PCR buffer that will be used within the screening. This can be done by amplifying any DNA sequence that contains the selected restriction site and verifying that the restriction enzyme digests the amplicon.

24. Incorporating a different restriction enzyme site in the second ssODN is useful for identifying clones in which both alleles have been targeted with the different ssODNs but is not essential.

25. When transfecting two ssODNs, use 20 pmol of each. Large amounts of the ssODNs can be toxic for the cells.

26. This protocol requires enzymatically passaged hiPSCs. For dissociation we use 1 × TrypLE Select enzyme and seed the cells at 3.75 × 10⁴ cells/cm² 48 h prior to transfection. However, the seeding density might vary with different cell lines. For each guide one well is prepared.

27. This is calculated based on 1.25 × the volume of Lipofectamine™ Stem Transfection Reagent required to correct for pipetting errors.

28. In our experience ifthe screen does not indicate that the pool of transfected cells contains targeted hiPSCs then we also do not obtain a correctly targeted clone following clonal isolation and screening. We would recommend performing the transfection procedure again using either an alternative transfection strategy or a different sgRNA.

29. Subcloning hiPSCs is most effective when the cells are in an exponential growth phase. If the cells are too confluent (>70%), cloning efficiency will likely be reduced.

30. Using a higher surface density of LN521 improves the likelihood that the single hiPSCs will attach. Later this is gradually decreased back to 0.5 μg/cm². The LN521-coated plates also can be prepared the day before single cell deposition and stored at 4 ° C overnight. However, they will still need to be incubated at 37 °C for 30 min prior to use. Typically, we obtain 15–30 colonies per 96-well plate and therefore from 4 plates we will obtain ~100 colonies for screening.

31. As there is an increased risk of contamination during the single cell deposition procedure using a flow cytometer we include Primocin, a broad-spectrum antimicrobial agent, rather than Penicillin–Streptomycin in our culture medium for the first 10 days after sorting.

32. It is critical that the LN521-coated wells do not dry out as the LN521 matrix becomes inactivated. Therefore, work quickly to remove the LN521 solution and replace with culture medium. Alternatively, half a 96-well plate can be processed at time.

33. Due to the increased risk of contamination, the single cell-sorted hiPSCs should be maintained separately from other cultures until they are confirmed to be mycoplasma negative. The laminar flow hood and other equipment used when maintaining the cells should be thoroughly cleaned with 1% SDS, 80% ethanol and exposed to ultraviolet light after each procedure. Also keep separate aliquots of all cell culture reagents used to maintain these cells.

34. To avoid unnecessary wastage of the culture medium, only refresh the marked wells.

35. Some colonies will not be viable when passaged. Ifthe colony appears to have stunted growth do not passage it.

36. In each 96-well plate do not seed cells in the last two wells (H11 and H12). These positions will be used for the positive and negative control samples when screening the clones.

37. When first undertaking this procedure, process a maximum of 10 wells simultaneously to prevent the harvesting taking too long and resulting in either the dissociated cells reattaching and/or the passaged cells having poor viability. An experienced operator can process up to 20 clones per round of dissociation.

38. It is possible to use 1x TrypLE Select Enzyme for this step. However, the dissociation solution then should be aspirated from the wells after 1½ min of incubation and before the cells have detached. The plate is then returned to the incubator without additional media. The enzyme that remains will continue to cleave the cell-cell junctions. After 5 min add culture medium and pipette up and down to generate a single cell suspension.

39. Passaging the colony at this ratio (~60:40 DNA:Archive) means the colonies on the DNA plate typically can be processed 1 day before the colonies on the Archive plate need to be cryopreserved. If you wish to try and complete the screening of the subclones prior to needing to process the Archive plate, then this ratio should be altered (e.g., 80:20 DNA:Archive). Any clones identified as being correctly targeted can then be maintained in culture. However, if the screening procedure takes longer than expected it might still be necessary to cryopreserve the colonies on the Archive plate. With the 60:40 DNA:Archive ratio, cells on the DNA plate are usually >90% confluent 2 days after passaging, while the cells on the Archive plate are ready for processing 3–4 days after passaging.

40. Use a multichannel pipette to prepare a separate 96-well plate with 1 μl of DNA loading dye per well. Pipette up and down the 5 μl of PCR product with the dye to mix and then use the same tip to load the samples into the agarose gel.

41. Always sequence with both the forward and reverse sequencing primers in case the trace from one of the sequencing reactions is of poor quality.

42. Work on ice and process all samples within 5 min to minimize DMSO toxicity.

43. Before transferring to liquid nitrogen storage drain any excess isopropanol from the bottom of the rack and blot dry on paper towels. Residual isopropanol will freeze in liquid nitrogen making it difficult to remove individual tubes from the rack later. Clones can be stored in this format for at least 12 months.

44. Try to remove as much of the mineral oil as possible, however any remnants will not affect the culture and can be removed the next day.

45. It is recommended to check the genomic integrity of any clones through karyotyping, SNP genotyping or whole genome sequencing. It is also possible that the clones may carry off-target mutations elsewhere in the genome. Genome sequencing can be used to screen for these although this can be costly. Alternatively, several different clones can be analyzed in any downstream assays. Finally, pluripotency and differentiation efficiency of the targeted clonal hiPSCs should be assessed.