Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants

Abstract

CRISPR/Cas9, base editors and prime editors comprise the contemporary genome editing toolbox. Many studies have optimized the use of CRISPR/Cas9, as the original CRISPR genome editing system, in substituting single nucleotides by homology directed repair (HDR), although this remains challenging. Studies describing modifications that improve editing efficiency fall short of isolating clonal cell lines or have not been validated for challenging loci or cell models. We present data from 95 transfections using a colony forming and an immortalized cell line comparing the effect on editing efficiency of donor template modifications, concentration of components, HDR enhancing agents and cold shock. We found that in silico predictions of guide RNA efficiency correlated poorly withactivity in cells. Using NGS and ddPCR we detected editing efficiencies of 5-12% in the transfected populations which fell to 1% on clonal cell line isolation. Our data demonstrate the variability of CRISPR efficiency by cell model, target locus and other factors. Successful genome editing requires a comparison of systems and modifications to develop the optimal protocol for the cell model and locus. We describe the steps in this process in a flowchart for those embarking on genome editing using any system and incorporate validated HDR-boosting modifications for those using CRISPR/Cas9.

Keywords: CRISPR; CRISPR/Cas9; cell line; genome editing; homology directed repair (HDR); prime editing; stem cells.

FIGURE 1

Workflow for early characterization of bulk populations by MiSeq followed by clonal line isolation by colony picking. (A) Design of CRISPR/Cas9 components. Five ssODN designs were compared. *: phosphorothioate bonds. Green “T” indicates PAM modification to prevent cleavage of the donor. (B) iPSCs were transfected under different experimental conditions and screened by MiSeq NGS. Populations with the highest rates of HDR were selected for colony picking. Individual colonies showing accurate repair by MiSeq were expanded as isogenic lines.

FIGURE 2

Comparison of HDR efficiency associated with protocol modifications at the bulk population level and editing outcomes in clonal lines. (A–B) Proportion of reads showing accurate repair by HDR in bulk population DNA transfected with (A) different ssODNs (p-value <0.45, Kruskal–Wallis test) and (B) different experimental conditions (p-value < 0.01, Kruskal–Wallis test). Asymmetric PAM: asymmetric donor without blocking mutation in PAM. Asymmetric PT: addition of phosphorothioate nucleotides to asymmetric ssODN. Asymmetric RC: reverse complement of asymmetric ssODN. HDR: addition of Alt-R™ HDR Enhancer after transfection. DMSO: addition of DMSO after transfection. NoHDR: no HDR enhancer or DMSO after transfection. (C) Editing outcomes in 100 colonies picked from two transfections showing population HDR rates of 11 and 12%.

FIGURE 3

Workflow for early single cell sorting and high throughput genotyping. (A) Design of CRISPR/Cas9 components targeting the G177D SNV in TBXT. (B) After transfection, U-CH1 bulk populations were screened by ddPCR. Populations showing HDR (2–10%) were single cell sorted using FACS. DNA was extracted from expanded clonal cell populations by establishing a “mirror plate” and used directly for genotyping by TaqMan™ qPCR or ddPCR (C) Bar plot of proportion of reads showing accurate HDR when concentrations of CRISPR/Cas9 components are varied. (D) Dot plots showing the gating strategy for sorting U-CH1 cells based on TOPRO3 and ATTO-550 fluorescence.

FIGURE 4

Proposed comprehensive flowchart for editing stem cells or immortalized cell lines using CRISPR/Cas9. *(Heigwer et al., 2014; Labun et al., 2019; Sledzinski, Nowaczyk and Olejniczak, 2020) and (Schubert et al., 2021) for HDR-specific ssODN design.