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A high efficiency precision genome editing method with CRISPR in iPSCs – Scientific Reports

We tested our approach (Fig. 1) on GWAS risk variant associated with tauopathy. The SNP variant rs867529 in the human EIF2AK3 (Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3) gene results in an amino acid change from serine to cysteine at amino acid position 136, by changing the nucleotide cytosine (C) to guanine (G) (Supplementary Fig. 1a). We identified a Protospacer Adjacent Motif (PAM) four nucleotides from the SNP and directed a DNA cut by Cas9 at the SNP. As previously reported, a homozygous Homology-Directed Recombination (HDR) event is best achieved when the guide RNA induces the cleavage less than ten nucleotides from the intended mutation11. We used single strand oligonucleotide (ssODN) as the repair template to introduce the desired editing for the homologous recombination. To avoid re-editing and improve editing efficiency, we introduced a silent mutation to the PAM site in the repair template11. We observed transfection with plasmid encoding shRNA (small hairpin RNA) against p53 improved the HDR rate to a mean of 30.8% by ICE (Inference of CRISPR Edits) analysis (Synthego), which was 11 times higher than the base protocol (Fig. 2a, b). When we added an HDR (homology-directed repair) enhancer (IDT), electrophoresis enhancers (IDT) and ​CloneR (STEMCELLTechnologies) to improve the cell survivability, the HDR increased to a mean of 59.5%, 21 times higher than the base protocol (Fig. 2a, b).

Figure 1
figure 1

Schematic diagram representing outline of study design. Editing was done by using CRISPR-Cas9 technology; base, modified and final protocols. Base protocol is defined as using only gRNA and ssODN. Modified protocol is defined as using shRNAp53 in gRNA and ssODN cocktail. Final protocol is defined by using ssODN, sgRNA (single guide RNA), shRNAp53, electroporation enhancer, CloneR and HDR enhancer.

Figure 2
figure 2

p53 inhibition and pro-survival factors improve editing efficiency (a), Percent HDR rate after including shRNAp53 and HDR enhancer, electroporation enhancers and CloneR in the base protocol for knocking in the SNP variant rs867529 in the human EIF2AK3 gene. (b), Fold change relative to base protocol after shRNAp53 and HDR enhancer, electroporation enhancer and CloneR in the base protocol SNP variant rs867529 in the human EIF2AK3 gene. (c), Percent HDR rate increase by including shRNAp53 and HDR enhancer, electroporation enhancer and Clone R in the base protocol in another SNP variant (rs13045) for the EIF2AK3 gene. n = 3 experiments with 1–3 biological replicates in each experiment. Error bars indicate standard deviation. Statistical analysis was performed by ANOVA with Tukey’s post-hoc analysis in (a and b), student’s t-test in (c). (d) Details of cell lines that were edited using the final protocol and their percent HDR rate.

We also tested this approach with another SNP variant (rs13045) for the EIF2AK3 gene, which changes the nucleotide at amino acid position 166 from guanine (G) to adenine (A), resulting in amino acid change from arginine (R) to glutamine (Q). We tested a Cas9 cleavage site which is four nucleotides away from the SNP. Using the base protocol, the HDR efficiency was 4%, but using the optimized final protocol, the HDR efficiency was increased to 25%. Here, we did not alter the PAM site, for alteration would introduce a missense mutation. The increase in HDR efficiency is about sixfold higher compared to the base protocol (Fig. 2c).

Next, we examined the efficiency of our final protocol in other iPSC lines. We applied the final protocol to introduce the APOE R136S Christchurch mutation in three different iPSC lines (PS 1.9.1, ADRC iPSC 5.3 cell line 1 and ADRC iPSC 5.3 cell line 2) (Fig. 2d). We observed 49% knock-in efficiency by ICE analysis in bulk sequencing in PS1.9.1 cells, and 100% knock-in in all of the subclones sequenced [(46.15% (12/26) Homozygous, 53.84% (14/26) Heterozygous]. Similarly, we observed 82–99% knock-in efficiency in ADRC 5.3 cell line 1 and 87% knock-in efficiency in ADRC 5.3 cell line 2 by ICE analysis. 100% and 94.4% of the subclones respectively have been edited. We also performed reverse mutation in the PS1.9.1 iPSC line to correct the PSEN1 E280A mutation using the final protocol. Comparably to other knock-ins, we observed 97 to 98% knock-in by ICE analysis in bulk sequencing and 100% knock-in after subcloning to single cell clones (Fig. 2d). Importantly, all the clones were found to be karyotypically normal by G-banding analysis (Supplementary Fig. 1b).

We performed whole genome sequencing (WGS) on PS1.9.1 APOE Christchurch edited cells to detect the gene editing status and any unwanted off target modifications. The WGS confirms the successful introduction of the Christchurch mutation. Analysis using DELLY12 for the detection of somatic large structural variations, revealed an inversion on chromosome7. However, no detectable off-target modifications were noted from the CRISPR Cas9 by using Cas-OFFinder software 13 (Supplementary Fig. 1c).