Journal: The CRISPR Journal

Article Title: Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins Results in High Levels of Gene Editing in Primary Hepatocytes

doi: 10.1089/crispr.2021.0134

Figure Lengend Snippet: On- and off-target Cas9 activity in electroporated Hepa 1-6 cells. (A) Schematic of chemical modifications in the single-guide RNA (sgRNA) structure. (B) On-target insertions/deletions (indels) for unmodified and chemically modified synthetic sgRNA co-transfected along with Cas9 protein. (C) On-target indels for Cas9 delivered as pDNA, mRNA, wild-type (WT) RNP, and HiFi RNP along with MS-modified sgRNA. (D) On- and off-target indels assessed using deep sequencing. Indels were evaluated using Tracking of Indels by Decomposition (TIDE) in (B) and (C) . Dots represent different electroporation experiments, and horizontal bars represent the means ( n = 3). * p < 0.05; ** p < 0.01. Mock samples were electroporated with pmaxGFP.

Article Snippet: The Hepa1-6 and HEK293 cell electroporation conditions were as follows: 20 μL SF nucleofection buffer; 1.2 × 10 5 cells; 0.5 μL of 20 μg/μL sgRNA; and 0.76 μL of 1.32 μg/μL Cas9 pDNA, 1 μL of 1 μg/μL CleanCap Cas9 mRNA (product code: L-7206-1000; TriLink Biotechnologies), 1.7 μL of 61 μM 3NLS SpCas9, or 1.7 uL of 61 μM 3NLS HiFi SpCas9 (product code: 1078728; Integrated DNA Technologies).

Techniques: Activity Assay, Modification, Transfection, Sequencing, Electroporation

Journal: The CRISPR Journal

Article Title: Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins Results in High Levels of Gene Editing in Primary Hepatocytes

doi: 10.1089/crispr.2021.0134

Figure Lengend Snippet: On- and off-target Cas9 activity in electroporated cryopreserved primary mouse hepatocytes. (A) On-target indels for unmodified and chemically modified sgRNA electroporated along with Cas9 protein. (B) Phase-contrast and fluorescence microscopy image of mouse hepatocytes electroporated with enhanced green fluorescent protein (eGFP) mRNA. The scale bar corresponds to 400 μm. (C) Comparison of on-target indels for primary hepatocytes electroporated with Cas9 mRNA, WT RNP, and HiFi RNP. (D) Indel frequencies in Hpd and three off-target sites in hepatocytes electroporated with Cas9 mRNA, WT RNP, and HiFi RNP. Indels were quantified using TIDE in (A) – (C) and deep sequencing in (D) . Dots represent different electroporation experiments, and horizontal bars represent the means ( n = 3). Mock samples were electroporated with eGFP mRNA.

Article Snippet: The Hepa1-6 and HEK293 cell electroporation conditions were as follows: 20 μL SF nucleofection buffer; 1.2 × 10 5 cells; 0.5 μL of 20 μg/μL sgRNA; and 0.76 μL of 1.32 μg/μL Cas9 pDNA, 1 μL of 1 μg/μL CleanCap Cas9 mRNA (product code: L-7206-1000; TriLink Biotechnologies), 1.7 μL of 61 μM 3NLS SpCas9, or 1.7 uL of 61 μM 3NLS HiFi SpCas9 (product code: 1078728; Integrated DNA Technologies).

Techniques: Activity Assay, Modification, Fluorescence, Microscopy, Comparison, Sequencing, Electroporation

Journal: The CRISPR Journal

Article Title: Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins Results in High Levels of Gene Editing in Primary Hepatocytes

doi: 10.1089/crispr.2021.0134

Figure Lengend Snippet: Analysis of on- and off-target Cas9 activity and viability in freshly isolated mouse hepatocytes after electroporation. (A) Timeline of experimental procedures. MTT assay and gDNA extraction were performed in separate wells. (B) Phase-contrast and fluorescence microscopy image of hepatocytes electroporated with eGFP mRNA. The scale bar corresponds to 400 μm. (C) Comparison of on-target indels for isolated mouse hepatocytes treated with Cas9 mRNA or WT Cas9 RNP. (D) Viability normalized to untransfected control following electroporation as determined by MTT assay. (E) Albumin levels in conditioned media normalized to untransfected control. (F) Percentage of indels at on-target and 10 off-target sites measured using deep sequencing. (G) Percentage of on-target frameshift mutations in hepatocytes electroporated with Cas9 mRNA and RNP. Dots represent different electroporation experiments, and horizontal bars represent the means ( n = 3). * p < 0.05. Mock samples were hepatocytes electroporated with eGFP mRNA.

Article Snippet: The Hepa1-6 and HEK293 cell electroporation conditions were as follows: 20 μL SF nucleofection buffer; 1.2 × 10 5 cells; 0.5 μL of 20 μg/μL sgRNA; and 0.76 μL of 1.32 μg/μL Cas9 pDNA, 1 μL of 1 μg/μL CleanCap Cas9 mRNA (product code: L-7206-1000; TriLink Biotechnologies), 1.7 μL of 61 μM 3NLS SpCas9, or 1.7 uL of 61 μM 3NLS HiFi SpCas9 (product code: 1078728; Integrated DNA Technologies).

Techniques: Activity Assay, Isolation, Electroporation, MTT Assay, Extraction, Fluorescence, Microscopy, Comparison, Control, Sequencing

Journal: The CRISPR Journal

Article Title: Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins Results in High Levels of Gene Editing in Primary Hepatocytes

doi: 10.1089/crispr.2021.0134

Figure Lengend Snippet: On- and off-target Cas9 activity and viability in electroporated human hepatocytes. (A) The first image shows untransfected primary human hepatocytes 24 h after plating. The second image shows plated primary human hepatocytes at 24 h after electroporation. (B) On-target indels for human hepatocytes electroporated with WT RNP and HiFi RNP. (C) Viability normalized to untransfected control as determined by MTT assay. (D) Albumin levels in conditioned media normalized to untransfected control cells. Dots represent different electroporation experiments, and horizontal bars represent the means ( n = 3). Mock samples were hepatocytes electroporated with eGFP mRNA.

Article Snippet: The Hepa1-6 and HEK293 cell electroporation conditions were as follows: 20 μL SF nucleofection buffer; 1.2 × 10 5 cells; 0.5 μL of 20 μg/μL sgRNA; and 0.76 μL of 1.32 μg/μL Cas9 pDNA, 1 μL of 1 μg/μL CleanCap Cas9 mRNA (product code: L-7206-1000; TriLink Biotechnologies), 1.7 μL of 61 μM 3NLS SpCas9, or 1.7 uL of 61 μM 3NLS HiFi SpCas9 (product code: 1078728; Integrated DNA Technologies).

Techniques: Activity Assay, Electroporation, Control, MTT Assay

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: ( a ) Schematic representation of the screen. Human embryonic kidney (HEK293T) cell line contains a reporter cassette expressing a selection marker, guide (sgGFP) and a mutant GFP-BFP protein. The line is transfected with a GFP repair oligo and an arrayed plasmid library containing ~450 DNA repair proteins that are fused to the C-terminus of Cas9WT. The homology-directed repair (HDR) editing efficiency for each fusion is defined by the percentage of cells with restored GFP function in each transfected well (measured by FACS). ( b ) Normalized GFP recovery values from two independent screens. Both experiments n = 3, each data point represents an average of value of all six replicas (n = 2 × 3), where each replicate was normalized either to the experimental average (blue dots) or to the plate average (orange dots). p-Values were calculated by one-way ANOVA test, where the mean of each triplicate is compared to the combined mean of all other triplicates from the screen. ( c ) Experiment average-normalized GFP recovery values for 52 fusions, chosen based on their performance in panel ( b ). In HEK293T n = 4, one independent biological experiment, in retinal pigment epithelium (RPE-1), n = 2, two independent biological experiments. Statistical significance is calculated as in ( b ). ( d ) Normalized GFP recovery values for 10 best-performing and five worst-performing Cas9 fusions from panel ( c ). Protein fragments are denoted based on the position in canonical transcript (i.e. SPIDR AA400-916 corresponds to a fragment of SPIDR protein which starts at amino acid 400 and ends at amino acid 916). ( e ) Schematic representation of the experiment shown in f . ( f ) Experiment average-normalized HDR editing for 31 fusions in four endogenous loci (ELANE, RNF2, Enh 4–1, and CTCF1) in HEK293T cells, quantified by droplet digital PCR. Polymerase fusions are marked with red pointers. n = 3, one independent biological experiment for each locus. Statistical significance is calculated as in ( b ). The raw data points are visible in – . ( j ) Replication fork schematics. For all experiments, Cas9 was delivered as plasmid, the repair template as single-stranded oligonucleotide, and the guide was expressed from the genome.

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Expressing, Selection, Marker, Mutagenesis, Transfection, Plasmid Preparation, Digital PCR

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: Data are the mean values, error bars represent ±S.D. Top illustrations depict the positioning of the CRISPR-Cas9 complex at the target locus. The red arrow shows the direction of the approaching polymerase. The same dataset is shown in and .

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: CRISPR

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: Data are the mean values, error bars represent ±S.D. Top illustrations depict the positioning of the CRISPR-Cas9 complex at the target locus. The red arrow shows the direction of the approaching polymerase. The same dataset is shown in and .

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: CRISPR

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: ( a–j ) The cells were electroporated with Cas9 mRNA and repair DNA. Homology-directed repair (HDR) and non-homologous end-joining (NHEJ) editing were measured with droplet digital PCR (ddPCR). n = 3, one independent experiment, bar denotes mean value, error bars represent ± SD. The dataset is also presented in . ( k–l ) Comparison between the ddPCR and NGS-based editing detection, for RNF2 site ( k ) and ELANE site ( l ). n = 3, one independent experiment, bar denotes mean value, error bars represent ± SD.

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Non-Homologous End Joining, Digital PCR

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: ( a ) Schematic representation of the experiment shown in b. ( b ) Normalized homology-directed repair (HDR) efficiency of the seven best-performing fusions in five endogenous loci in hTERT immortalized fibroblasts (BJ-5ta), quantified by droplet digital PCR (ddPCR). Heat maps represent values normalized to Cas9WT for each gene locus. The sets have an upper limit threshold and values above it are color-coded as a scale maximum (bright-red). One independent experiment for each gene locus, n = 4 for FANCF, Enh 4–1, RNF2, ELANE gene loci; n = 3 for STAT3 gene. The raw data points are visible in . For all experiments, Cas9 was delivered as mRNA, the repair template as single-stranded oligonucleotide, and the guide was expressed from the genome.

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Digital PCR

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: ( a ) Schematic representation of experiment shown in b. ( b ) The DNA repair progression in the retinal pigment epithelium (RPE-1) reporter locus for eight of the better-performing fusions (selected from screens on ). Droplet digital PCR (ddPCR) quantification, n = 3, single independent experiment, bar denotes mean value, error bars represent ± SD. Orange highlighting indicates the Cas9WT homology-directed repair (HDR) editing level at 24 hr post-electroporation. p-Values denote significance of the total editing (HDR+ non-homologous end-joining [NHEJ]) increment between the Cas9WT and other fusions (for 24–48 hr period). Statistical values derived using one-way ANOVA test. Cas9 was delivered as plasmid, the repair template as single-stranded oligonucleotide, and the guide was expressed from the genome. ( c ) Schematic representation of experiment shown in d–i . ( d ) Representative immunofluorescence image used for DNA breakpoint quantification. Cell nuclei are depicted in blue, γH2AX foci in green, and red arrows indicate individual foci. Scale bar 50 µm. ( e ) Time course for γH2AX foci emergence in human immortalized fibroblasts (BJ-5ta). The cells were electroporated with Cas9WT or Cas9 fusion to DNA polymerase delta subunit 3 (Cas9-POLD3) mRNA and a pool of three guides targeting RNF2, Enh 4–1, and STAT3 loci. The CTCF1 gRNA is constitutively expressed. Five confocal images were taken for each condition, n = 5, single independent experiment, bar denotes mean value, error bars represent ± SD. Statistical significance of the difference between Cas9WT and Cas9-POLD3 is calculated using ANOVA test for the equality of the means at a particular time point. ( f–i ) ddPCR quantification of the NHEJ repair dynamics across time for the experiment described in ( e ). The red arrow shows the direction of the approaching polymerase. The DNA strand which the CRISPR-Cas9 binds to is colored in red. n = 1, single independent experiment, bar denotes mean value, error bars represent ± SD. Cas9 was delivered as mRNA, the repair template as single-stranded oligonucleotide, one guide was expressed from the genome and three guides transfected as synthesized oligonucleotides.

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Digital PCR, Electroporation, Non-Homologous End Joining, Derivative Assay, Plasmid Preparation, Immunofluorescence, CRISPR, Transfection, Synthesized

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: ( a ) Schematic representation of the affinity purification mass spectrometry (AP-MS) experimental workflow. ( b ) Protein interaction map of the Cas9 fusions and Cas9WT. The interacting endogenous proteins are clustered into circles based on how many Cas9 proteins they interact with. The endogenous proteins in the bottom left circle interact with only one fusion or Cas9WT, and the proteins in the bottom right circle interact with all the fusions or Cas9WT. Pink coloring represents RNA-binding proteins, light-blue represents histone proteins, dark blue indicates interaction partners with helicase activity, and light blue circles denote proteins with other functions. Each AP-MS experiment was conducted in three biological replicates (n = 3). The Cas9 and gRNA were expressed from the genome.

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Affinity Purification, Mass Spectrometry, RNA Binding Assay, Activity Assay

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: ( a ) Peptide-spectrum match (PSM) counts of the Cas9 fusion proteins Flip-in human embryonic kidney (HEK293T) cell lines after 30 hr tetracycline induction. The samples were subsequently used in affinity purification mass spectrometry . ( b ) Functional activity of the CRISPR-Cas9 fusions used in the mass spectrometry assays. Expression of the fusion construct was induced with tetracycline 24 hr prior to the transfection with ribonucleoprotein (RNP) complex targeting GFP locus and corresponding repair template. GFP recovery evaluated by fluorescence-activated cell sorting (FACS) after 5 days.

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Affinity Purification, Mass Spectrometry, Functional Assay, Activity Assay, CRISPR, Expressing, Construct, Transfection, Fluorescence, FACS

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: ( a ) Mismatch plot of the GUIDE-Seq results for Cas9WT and Cas9-POLD3, using a published guide against the endogenous human embryonic kidney (HEK) site 4. Cas9 and the guide were co-transfected as plasmids. The on-target sequence is depicted at the first line of the table. The most abundant off-targets are listed underneath the intended edit and sorted by frequency, which is determined by dividing the number of reads that contain the off-target edit with the total read count. Gray lines connect the shared off-target sites. ( b ) Venn diagram of the common and unique off-target sites between Cas9WT and Cas9-POLD3. ( c–f ) Mismatch plots of the indel profiles of Cas9WT and Cas9-POLD3 in BJ-5ta fibroblasts, obtained by deep amplicon sequencing. Cas9 was delivered as mRNA, the repair template as single-stranded oligonucleotide, and the guide was expressed from the genome. The on-target gRNA-binding site is on the top row, highlighted with a black box. The most frequent indels are below, sorted according to their frequency. The indel frequency is calculated by dividing the indel read count by the sum of the top 10 indel read counts. The edits matching the homology-directed repair (HDR) template are marked in red in the summary plots. Plots depict: ( c ) On-target editing signature of Cas9WT, RNF2 locus. ( d ) On-target editing signature of Cas9WT, ELANE locus. ( e ) On-target editing signature of Cas9-POLD3, RNF2 locus. ( f ) On-target editing signature of Cas9-POLD3, ELANE locus. ( g–h ) The most common non-homologous end-joining (NHEJ) and HDR editing outcomes depicted in bar graphs. n = 3, bar denotes mean value, error bars represent ± SD. Statistical significance is calculated with unpaired, two-sided Student’s t-test.

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Transfection, Sequencing, Amplification, Binding Assay, Non-Homologous End Joining

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: Retinal pigment epithelium (RPE-1) cells were electroporated with Cas9WT and Cas9-POLD3 plasmids along with an sgRNA targeting RNF2 locus. The RNA was extracted at the indicated time points (12, 24, 48, 72 hr) and CDKN1A expression level quantified by droplet digital PCR (ddPCR), with GAPDH as the normalization control. Heat map ( a ) values represent average of three replicates (n = 3, biological triplicates from one independent experiment). Bar graphs ( b ) show individual values from ( a ), bar denotes mean value, error bars represent ± SD. Statistical significance is calculated with unpaired, two-sided Student’s t-test (p-values on plot) and ANOVA .

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Expressing, Digital PCR

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: ( a ) Editing of GFP in reporter human embryonic kidney (HEK293T) cells, n = 4, one of two independent experiments, bar denotes mean value, error bars represent ± SD. Statistical significance is calculated with unpaired, two-sided Student’s t-test. Cas9 was delivered as plasmid, the repair template as single-stranded oligonucleotide, and the guide was expressed from the genome. ( b ) Normalized to Cas9WT HDR efficiency of the fusions in four endogenous loci (CTCF1, ELANE, Enh 4–1, RNF2) in HEK293T, hTERT immortalized fibroblasts (BJ-5ta) and RPE-1 cells quantified by droplet digital PCR (ddPCR). Cas9 was delivered as plasmid (HEK293T and RPE-1) or mRNA (BJ-5ta), the repair template as single-stranded oligonucleotide, and the guide was added in electroporation reaction (HEK293T and RPE-1) or expressed from the genome (BJ-5ta). Heat maps represent HDR values normalized to experimental average for each gene locus. Each cell type was tested in a single biological experiment (n = 3 HEK293T [top dataset] and BJ-5ta; n = 4 HEK293T [bottom dataset] and RPE-1). ( c ) GFP reporter locus editing efficiency of recombinant Cas9 fusion proteins in RPE-1 and HEK293T cells. n = 5, one of two independent experiments, bar denotes mean value, error bars represent ± SD. Statistical significance is calculated with ANOVA that is adjusted for multiple comparisons.

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Plasmid Preparation, Digital PCR, Electroporation, Recombinant

Journal: eLife

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.7554/eLife.75415

Figure Lengend Snippet: ( a ) hESCs and ( b ) PBMCs were electroporated with Cas9WT and Cas9-POLD3 mRNA, sgRNA targeting the RNF2 locus, and repair DNA. Homology-directed repair (HDR) and non-homologous end-joining (NHEJ) editing were measured with droplet digital PCR (ddPCR). n = 3, one independent experiment, bar denotes mean value, error bars represent ± SD. Statistical significance is calculated with unpaired, two-sided Student’s t-test.

Article Snippet: For each reaction, 0.4 × 10 6 of cells were resuspended in 25 µl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Non-Homologous End Joining, Digital PCR