Abstract
Genome editing with CRISPR-Cas9 of primary human cells is a powerful tool to study gene function. For many cell types there are efficient protocols for editing with optimized plasmids for Cas9 and sgRNA expression. Vascular cells, however, remain refractory to plasmid-based delivery of CRISPR machinery for in vitro genome editing. This is due to the low efficiency of transfection, poor expression of the Cas9 machinery, and the toxic effects of selection antibiotics. Here, we describe a method for high efficiency editing of primary human vascular cells in vitro using nucleofection to deliver sgRNA:Cas9-NLS ribonucleoprotein complexes directly. This method is more rapid, and its high editing efficiency eliminates the need for additional selection steps. The edited cells may then be employed in diverse applications such as gene expression measurement or functional assays to assess various genetic perturbation effects in vitro. This method proves effective in vascular cells that are refractory to standard genome manipulation techniques that use viral delivery of plasmids. We anticipate that this technique will be applied to other non-vascular cell types that face similar barriers to efficient genome editing.
Keywords: Genome editing, vascular smooth muscle cells, endothelial cells, CRISPR, Cas9
INTRODUCTION:
CRISPR-Cas9 genome editing is a powerful tool in molecular biology and is now being employed in the treatment of disease (Frangoul et al., 2021; Gillmore et al., 2021). Worldwide, cardiovascular disease has the highest burden of mortality, and progress in the research of cardiovascular disease may be accelerated via a rapid technique for CRISPR editing in the relevant cell types. Primary human vascular cells, particularly vascular smooth muscle cells (VSMCs), are notoriously refractory to genetic manipulation with typical cellular methods such as lipid-based transfection and typically require labor-intensive viral transduction. Such methods are limited by low transfection efficiency, potential cellular toxicity from antibiotic selection, and long experimental duration from multiple cloning steps to generate the plasmids encoding the CRISPR machinery.
Here, we describe a method for high-efficiency genome editing with direct delivery of CRISPR single-guide RNAs (sgRNAs) complexed with S. pyogenes Cas9-nuclear localization signal (NLS) protein as a ribonucleoprotein (RNP) in vascular cells. This method has multiple advantages. First, it allows for rapid screening of guides. Since the stabilized sgRNA molecules are commercially synthesized, it bypasses the normal requirement for multiple cloning steps to generate plasmids containing the guide sequence and/or virus packaging. Second, editing efficiency is significantly higher than lipid- and virus-based methods (without antibiotic selection). Third, it does so with minimal toxicity to cells, which continue to grow after nucleofection. Fourth, it bypasses the risks to the experimenter associated with the generation of live virus.
The methods below describe the appropriate reconstitution of guides, preparation of cells for nucleofection, the generation of sgRNA:Cas9 RNP complexes, and subsequent assessment of genome-editing with PCR. With these methods, we have had success in achieving gene knockouts or edits to the noncoding genome with high efficiency in the vascular cell types described. A variation of this method has been applied to human hematopoietic stem cells (Bick et al., 2020). In this protocol, we review a highly efficient method to edit primary human vascular cells with sgRNA:Cas9 RNP complex nucleofection that does not require the generation of viral vectors or antibiotic selection. We do not review the design of CRISPR guides, which is extensively reviewed elsewhere (see Internet Resources). Our protocol is optimized to achieve highly efficient (>80%) editing of any genetic locus in primary vascular cells, which can be used for downstream transcriptional or functional analysis.
CAUTION:
Follow all appropriate guidelines and regulations for the use and handling of human cells and tissues provided by your institution. All experimental procedures were conducted in accordance with the institutional review board-approved protocols at our institutions.
STRATEGIC PLANNING:
CRISPR guide design
Proper guide design is essential to the success of this protocol. We design guides via algorithms on publicly and commercially available software for managing genetic data, however multiple resources are available for guide optimization based on computational parameters. In general, we do not select guides that have a low on-target scores and have found drop-off in cutting efficiency below a rating of 60% (Doench et al., 2016; Hsu et al., 2013).
PCR primer design & PCR optimization
For the proper assessment of genome editing, it is necessary to generate a single amplicon of roughly 300-1000bp surrounding the desired genome edit(s). This entails primer design, optimization of polymerase choice and electrolyte conditions, as well as annealing temperature with the specific polymerase employed. As with CRISPR sgRNA design, multiple online resources are available for optimization of PCR primers.
BASIC PROTOCOL 1
CRISPR-Cas9 Genome Editing of Primary Human Vascular Cells In Vitro
The overarching aim of the technique is to introduce sgRNA:Cas9 ribonucleoprotein complexes into cells such that genome editing may occur. Nucleofection is a high efficiency method to achieve delivery of the sgRNA:Cas9 complex. If conducted properly, the user should be able to harvest cells 1-2 days after editing with efficiencies that exceed 80% per guide sequence. Cells may be maintained in culture for future applications with durable edits, provided there is no diminished fitness of cells as a consequence of the genomic edit. All typical precautions should be taken for the handling of primary human or animal cells in cell culture.
Materials:
Cell type of interest: Endothelial cells, vascular smooth muscle cells, or monocytes
Cell culture consumables: plates, flasks, culture media, trypsin solution, phosphate-buffered saline solution
Cell counter: Hemocytometer or equivalent
sgRNA guides (We use CRISPRevolution sgRNAs from Synthego, however custom synthetic sgRNAs are available from multiple vendors)
Synthetic Cas9-NLS protein (We obtain from Synthego, however also available from multiple vendors)
Nucleofection kit including nucleovette and solutions: SG Cell Line 4D-NucleofectorTM X Kit S (Catalog #: V4XC-3032)
Genomic DNA extraction kit
Optimized DNA oligonucleotide PCR primers for locus of interest
DNA polymerase
PCR consumables
DNA gels (1-2% agarose, ethidium bromide/sybr stain), TAE buffer
PCR purification kit
BSLII cell culture hood
Tissue culture microscope (Nikon or equivalent)
Benchtop centrifuge
Lonza 4D Nucleofector Core Unit (AAF-1002B)
Lonza 4D Nucleofector X Unit (AAF-1002X)
DNA gel electrophoresis tank
Sanger sequencing method
Protocol steps with step annotations:
sgRNA Guide Reconstitution (Figure 1A)
Figure 1. Overview of experimental procedure.
Graphical summary of the steps in this protocol. Steps A to E generally require less than two hours. Steps preceding A (namely, cell culture, guide design and primer design) and and follow-up experiments to address the effect of genomic perturbation are not addressed. Created with Biorender.
Briefly spin down sgRNAs in a benchtop microcentrifuge to collect all sgRNA to bottom of tube
Add sterile TE buffer for a final concentration of 50μm (e.g., add 60μl to 3.0 nmol of sgRNA)
Vortex at moderate-to-high intensity for 30 seconds to resuspend
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Briefly spin down and maintain on ice.
Note: sgRNAs are synthetically modified to be more stable relative to standard RNA molecules, and we observe no decrement to function with serial freeze-thaw cycles, however we minimize the time that guides are maintained at room temperature throughout this process. We store guides at −20°C
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Briefly spin down Cas9-NLS solution and maintain on ice.
Note: We store Cas9-NLS solution at −20°C, also taking care to minimize the time that this solution is at room temperature. Maintain on ice throughout the experiment.
Cell Preparation (Figure 1B, C)
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6
Prepare well plates with typical volume of culture media and place in 37°C incubator for cell recovery after nucleofection. Per reaction, prepare at least one well to assess genome editing efficiency plus any additional wells for parallel experiments (such as gene expression or functional assays).
Note: These wells will be used to rest cells immediately after nucleofection. In order to verify the effect of a sgRNA:Cas9 nucleofection reaction, we generally split one reaction immediately after nucleofection into two wells: one for DNA extraction to assess editing efficiency and one for RNA extraction or a functional assay. Thus, when cells are harvested contemporaneously, we have a precise assessment of editing efficiency. This is particularly important if there might be a fitness effect to the CRISPR genomic edits, which might differ in population frequency if the cells are harvested at different time points.
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7
From your primary cell culture, prepare a suspension of cells according to usual protocols for your cell type. For our primary vascular cells, this usually entails the application of 0.25% Trypsin-EDTA for 1-2 min after a PBS wash to sub-confluent cells, which have been in culture for 2+ days. Culture media containing serum is used to inhibit trypsin.
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8
Pellet cell suspension (5 min at 300 x g), decant supernatant carefully so as not to disrupt pellet, and resuspend with at least 1ml of PBS to wash off culture media & trypsin.
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9
Count cells and isolate a total volume of suspension containing 1.5-2.0 x 105 cells per reaction for the total number of reactions
(Example: calculation for 12 reactions (6 experimental & 6 control): 1.5 x 105 x 12 = 1.8 x 106 cells total)
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10
Pellet the number of cells needed (5 min at 300 x g) from PBS suspension and remove supernatant, taking care not to disturb pellet. Maintain cell pellet on ice until ready for use.
Note: Attempt to minimize the amount of remaining PBS, as excess PBS will diminish nucleofection efficiency
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11Prepare sufficient volume of sgRNA:Cas9-NLS complexes as a master mix.
- Per nucleofection reaction and desired double strand break, add to a PCR tube:
- 1μl Cas9-NLS solution (20 pmol)
- 1μl sgRNA (50 pmol) guide reconstituted as described above in Step 2
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For example, if 6 control and 6 experimental reactions are desired, add 6μl Cas9 and 6μl control sgRNA solution to control tube; then add 6μl Cas9 and 6μl of experimental sgRNA solution to the experimental tube.Note: Importantly, if multiple guides are used in the same reaction, mix guides before the addition of Cas9 so as to prevent unequal stoichiometry of sgRNA:Cas9 complex between guides.
- Mix well by pipetting up and down ~30x or briefly vortex and spin down
- Incubate at room temperature (allowing sgRNA:Cas9 RNP complexes to form) for at least 10 min, but not more than 30 min.
- Maintain stocks on ice or return to −20°C freezer after preparing sgRNA:Cas9 RNP solution
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12During centrifugation steps, prepare sufficient supplemented nucleofection solution for desired number of reactions.
- For example, 12 reactions = 240μl of total nucleofection solution. 3.6μl of SG supplement and 16.4μl of SG solution totaling 20μl per reaction.
Nucleofection Reaction and Re-plating of cells (Figure 1D, E)
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13
After sgRNA:Cas9 complexes have complexed for >10 min, resuspend cell pellet in supplemented SG solution (20μl per reaction, from step 11). Work quickly to minimize the time that cells are resuspended in SG.
Note: The single pellet will be split between experimental conditions in the next step, so the entire volume of supplemented SG solution should be used to resuspend the total cell pellet. (Example: If 6 control and 6 experimental reactions are desired, we prepare 240μl of SG solution to resuspend cell pellet, then add 120μl of cell solution to control sgRNA:Cas9 and 120μl of cell solution to experimental sgRNA:Cas9 RNP complex).
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14
Add 20μl of SG-cell suspension per desired number of reactions to complexed sgRNA:Cas9. Change pipette tips between experimental conditions to prevent cross-contamination of sgRNA:Cas9 complexes. Mix well by pipetting up and down
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15
Working quickly, add 22μl of cell/sgRNA:Cas9 suspension (20μl cell suspension + 1μl Cas9 + 1μl [or more] sgRNA) to each nucleovette well, taking care not to introduce bubbles (bubbles interfere with nucleofection efficiency).
Note: If a bubble develops in your nucleovette well, it may be removed by tapping the chamber on the cell culture hood surface or removed by sweeping up the wall of the well with a pipette tip
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16Insert nucleovette chamber into nucleofector in the proper orientation
- Select solution SG
- Select desired wells
- Program nucleofector based on cell type
- For endothelial cells: CA-167, CA-210
- For vascular smooth muscle cells: CM-137
- For THP-1 Cells: FF-100
- Run the nucleofection reaction
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17
Working quickly, add 100μl of warm culture media from prepared culture plate to each nucleovette well. The recoverable volume from each well volume should be ~115μl after this addition.
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18
Partition media containing cell suspension from each well for assessment of genome editing efficiency, gene expression, or functional assays, as desired.
Note: Cells should be allowed to rest overnight in growth conditions after this point
Assessment of Genome Editing Efficiency (Figure 1F)
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19
To assess genome editing, extract genomic DNA from edited cells as early as the following day. We use commercially available resin column kits, however traditional phenol-chloroform extraction may also be used
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20
Amplify the DNA segment containing the sgRNA targets with PCR
Note: We design PCR primers such that amplicons have lengths of ~500-800 bp. This makes it possible for varying size deletions with dual guides to be assessed after a single PCR primer pair has been optimized for a genomic locus.
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21
To assess efficiency of a single guide strategy, perform PCR cleanup and send for Sanger sequencing (Figure 2A). Use ICE or TIDE per Online Resources below (Brinkman et al., 2014; Hsiau et al., 2018).
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22
To assess efficiency of dual guide strategy for excision of a genomic segment by nonhomologous end-joining, run the PCR product on a 2% DNA gel (or higher for smaller deletions <100 bp) and image on in UV-safe imaging setup or comparable imaging method, ensuring that bands are not over saturated. Take care to confirm that amplicon sizes are comparable with expected lengths as related to DNA ladder (Figure 2B).
Note: Ratio of native and edited band intensity may be quantified using ImageJ (NIH) to provide a relative estimate of editing efficiency (Bick et al., 2020).
Figure 2. Assessment of editing efficiency.
(A) Sanger sequencing of locus targeted by sgRNA sequence at high efficiency with targeting guide (upper panel) and control guide (lower panel) exhibiting a significant decrement to signal quality after CRISPR-Cas9 mediated double-strand break and nonhomologous end-joining. (B) Schematic of possible outcomes with two double strand breaks from a two-guide strategy; created with Biorender. (C) DNA gel demonstrating efficient deletion of a 65bp segment of DNA using a paired guide strategy with nonhomologous end-joining of two CRISPR cuts. Amplicon without deletion (blue) appears the same length as the control band. Note the greater intensity of the band with deletion, signifying highly-efficient deletion.
COMMENTARY:
Background Information:
We developed this protocol given the need for a robust and rapid editing strategy for cells pertinent to the cardiovascular system. The central advantages of this technique are that it affords high efficiency, rapid screening of guides, rapid experimental readouts, and does so without toxic antibiotic selection. These features are discussed at greater length in “Editing efficiency” below.
We have employed this method to perturb both the coding and noncoding genome in small scale, high biological replicate experiments wherein our readouts have been gene expression and functional assays in cellular biology. This technique may be further applied to long term expansion of larger numbers of cells, and as such, will likely be useful in cell-engineering applications.
Of note, we describe the application of this technique to double-strand breaks with S. pyogenes Cas9 protein, wherein such breaks are repaired by nonhomologous end-joining. This technique may also be applied to the generation of double-strand breaks with homology-directed repair; however, a discussion of this technique is beyond the scope of this protocol.
Critical Parameters:
Editing Efficiency
In general, we observe editing efficiencies >80% by ICE or TIDE computational analysis per sgRNA across cell types as soon as 18 hours after the experimental procedure. This far exceeds the efficiency of the same guide sequences employed in plasmid-based transfection (<10% in ECs, undetectable in THP1 monocytes and VSMCs) or nucleofection of these plasmid-based vectors (10-20% in THP1 monocytes, 20-30% in endothelial cells, undetectable in VSMCs) in our hands. This efficiency also exceeds that of lentiviral methods without antibiotic selection (60-70%) in our hands.
Editing efficiency reflects a composite endpoint of multiple experimental factors that include guide efficacy, technical factors (excessive PBS or unremoved culture media, exposure time to nucleofection solution), optimization of nucleofection program (for both viability and editing), fitness effects of any edits, duration between nucleofection and harvest if there are fitness effects, and PCR quality. Additionally, if a multiple guide strategy is employed, we hypothesize that greater distances between guides will result in a higher frequency of local indels at the targeted sites than excision of the genomic segment between the guides. We have observed high efficiencies of excision of genomic segments of up to 550bp.
This method does not allow for antibiotic selection of editing cells with an antibiotic resistance gene. However, we observe editing efficiencies that are high enough such that the benefits of higher editing efficiency are outweighed by the drawbacks of toxic antibiotic exposure and prolonged experimental durations. If higher efficiencies are desired with this method, we surmise that serial rounds of nucleofection with the target sgRNA:Cas9 RNPs and culture may produce a greater purity of edited cells.
In general, we feel that cells should be harvested for DNA and assessment of genome editing efficiency contemporaneously with other experimental readouts so that the experimenter will know the strength of the genomic perturbation involved in the experimental results. For us, this has meant that we extract DNA from each biological replicate at the same timepoint that we extract RNA or perform a functional assay and report data on editing and other readouts together.
Cell Numbers
We have optimized the use of this protocol for small volumes of cells in the small size nucleofection chamber. Larger chambers exist for the nucleofection of higher cell numbers. We have also tested the editing efficiency of vascular endothelial cells at 2 x 106 cells per nucleofection reaction using the Lonza SG Cell Line 4D-Nucleofector X Kit L (Catalog #: V4XC-3024). This larger nucleovette system uses 100 μL of nucleofection solution. We were able to achieve editing efficiency comparable to the 20 μL reaction described above by scaling up Cas9 and sgRNA volumes by 5-fold and using the same nucleofection program. The ability to edit larger cell numbers was particularly useful for functional assays that required more cells.
Optimization of Nucleofection program
We observed that not all nucleofection programs suggested by the manufacturer in their protocols were effective for efficient nucleofection and preservation of cell health and viability (see “Online Resources” for nucleofection protocols from the manufacturer). We recommend optimization of the nucleofection program using a control plasmid before proceeding to nucleofection with sgRNA:Cas9 RNP. This may be most rapidly undertaken with the use of the pMaxGFP vector that is supplied with the nucleofection kit and subsequent live-cell fluorescence microscopy for assessment of both expression and cell viability. Cells should express green fluorescent protein within 24 hours after nucleofection. We found that efficiency of pMaxGFP nucleofection paralleled efficiency of sgRNA:Cas9 RNP editing efficiency for each program. After screening the programs suggested by the manufacturer for each cell type, further optimization of the programs may be undertaken by contacting technical support as outlined in nucleofection protocols (see “Online Resources” for nucleofection protocols from the manufacturer).
Of note, primary human vascular cells are resistant to efficient transfection with lipid-based methods for other molecules, such as siRNAs. We have observed that siRNAs may be substituted in this protocol in place of sgRNA:Cas9 complexes to achieve high efficiency transcript knockdown using identical nucleofection programs as above.
Troubleshooting:
Understanding Results:
The primary aim of this method is to perform high efficiency genome editing with rapid testing of guides such that edited cells may be used in downstream applications, consistent with the desired aims of the user. The central data generated from this protocol is evidence of genome editing, which may be accomplished in different ways depending on the sgRNA targeting strategy.
Cas9 generates double strand breaks (DSBs) in the DNA that can be repaired either by nonhomologous end-joining (NHEJ), which results in the introduction of insertion-deletion mutations, or homology directed repair. To assess the efficiency of an individual double-strand break and subsequent repair by NHEJ, a PCR amplicon containing the targeted sequence should be generated and sequenced with Sanger sequencing, with subsequent TIDE or ICE analysis in comparison with a control specimen, or Next-Gen sequencing, that will more precisely quantify variant allele fractions because of DSBs. An abrupt drop-off in the quality of Sanger sequencing 2-3bp from the protospacer adjacent motif will indicate a strong CRISPR DSB and subsequent repair by NHEJ (Figure 2A) with multiple possible outcomes (Figure 2B). We have also observed that if the sgRNA is particularly effective and the majority of NHEJ results in a specific insertion-deletion mutation, the Sanger sequencing quality will be relatively preserved with a clear novel dominant amplicon in the Sanger sequencing data. TIDE and ICE are computational methods that provide a quantitative estimate of the fraction of edited amplicons, as well as an estimate of the populations of precise indel mutations contained within the population as a consequence of NHEJ (Brinkman et al., 2014; Hsiau et al., 2018).
This analysis may not be conducted to assess the efficiency with which a segment of DNA was deleted, such as in a strategy with multiple sgRNAs and DSBs are achieved, wherein NHEJ joins two previously nonadjacent DNA segments. For a quantitative assessment of this phenomenon, Next-Gen sequencing may be employed, which is beyond the scope of this protocol. Another method that provides an estimate of frequency of DNA segment deletions is to provide a relative quantification of amplicon intensity between native and segment-deleted bands (Figure 2B). This may be accomplished with DNA gel electrophoresis and gel analysis with ImageJ (National Institutes of Health) or similar image analysis software (Bick et al., 2020).
Time Considerations:
The initial phases of the experimental protocol from guide reconstitution to nucleofection of sgRNA:Cas9 RNP complexes typically takes less than two hours for a moderate scale experiment. Further time considerations regard the timing of cell preparation from a culture and the timing of subsequent experimental conditions after genome editing. As noted above, highly efficient genome editing is observed as rapidly as 18 hours after nucleofection. Timing may be abbreviated by preparing guide complexes in parallel with centrifugation steps, rather than in series.
sgRNAs and Cas9 protein are temperature sensitive, and so these reagents should not be subjected to prolonged exposure to room temperature. We have observed no decrement to function with guide complexes after serial freeze-thaws, given that these sgRNA molecules have chemical modifications for stabilization, however we recommend minimizing freeze-thaw cycles to the extent possible by aliquoting.
Finally, we recommend minimizing the time that cells are exposed to nucleofection solution. We observe that prolonged exposure to this solution is toxic to cells. We recommend resuspension of the cell pellet in SG nucleofection solution immediately before aliquoting the cell suspension into experimental conditions and working quickly through the nucleofection steps, such that cells are replated to culture conditions at 37°C as rapidly as possible.
Table 1.
Troubleshooting Guide for DNA Blotting and Hybridization Analysis
Problem | Possible Cause | Solution |
---|---|---|
Low Editing Efficiency | Ineffective guides | Guide redesign with higher on-target score |
Degraded guides/Cas9 | New reagents | |
Suboptimal nucleofection program choice | Optimization of nucleofection program for viability and editing | |
Excessive media/PBS | Failure to wash cells with PBS before resuspension in nucleofection solution or excessive PBS with pellet upon resuspension. | |
Cells with diminished fitness as a consequence of intended genome edit | More rapid assessment of genome-editing after nucleofection. | |
Excessive cell death | Genomic manipulation of a locus essential for viability | Reconsideration of experimental design in light of biological consequence of manipulation |
Prolonged exposure to nucleofection solution | Working more rapidly through steps that expose cells to nucleofection solution | |
Nucleofection program resulting in lethality | Optimization of nucleofection program for viability and editing |
ACKNOWLEDGEMENTS:
The authors acknowledge Richard Voight MD, PhD and Vijay Sankaran MD, PhD for guidance.
Support for DSA,VSL-K, and MY provided by NIH 5T32HL7604-35. Work in the lab of RMG is funded by NIH 1DP2HL152423-01.
Footnotes
CONFLICT OF INTEREST STATEMENT:
Authors declare no conflicts of interest.
DATA AVAILABILITY STATEMENT:
Data available upon request.
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INTERNET RESOURCES:
URLs for important sites relevant to the method. Each must be accompanied by a short description of the subject of the site.
We find the following resources to be valuable for guide design:
https://zlab.bio/guide-design-resources
We found the following manufacturer guidelines helpful in the optimization of this protocol:
Amaxa™ 4D-Nucleofector™ Basic Protocol for Primary Mammalian Smooth Muscle Cells For 4D-Nucleofector™ X Unit–Transfection in suspension: https://bioscience.lonza.com/download/content/asset/21660
Amaxa™ 4D-Nucleofector™ Basic Protocol for Primary Mammalian Endothelial Cells for 4D-Nucleofector™ X Unit–Transfection in suspension: https://bioscience.lonza.com/download/content/asset/21684
Amaxa™ 4D-Nucleofector™ Protocol for THP-1 [ATCC®] For 4D-Nucleofector™ X Unit – Transfection in suspension https://bioscience.lonza.com/lonza_bs/US/en/document/21575
Synthego CRISPR Resources: https://www.synthego.com/resources/all/protocols
Online methods to quantify insertion-deletion mutations as a result of CRISPR genome edits, as discussed in (24) above.
Inference of CRISPR Edits: ICE analysis (Hsiau et al., 2018).
https://www.synthego.com/products/bioinformatics/crispr-analysis
Tracking Indels by Decomposition: TIDE (Brinkman et al., 2014).
ImageJ (NIH)
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data available upon request.