Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants

Inga Usher; Lorena Ligammari; Sara Ahrabi; Emily Hepburn; Calum Connolly; Gareth L Bond; Adrienne M Flanagan; Lucia Cottone

doi:10.3389/fgeed.2022.932434

Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants

Front Genome Ed. 2022 Jul 5:4:932434. doi: 10.3389/fgeed.2022.932434. eCollection 2022.

Authors

Inga Usher¹, Lorena Ligammari¹, Sara Ahrabi², Emily Hepburn³, Calum Connolly³, Gareth L Bond⁴, Adrienne M Flanagan^{1

5}, Lucia Cottone¹

Affiliations

¹ Department of Pathology (Research), UCL Cancer Institute, University College London, London, United Kingdom.
² Department of Haematology, UCL Cancer Institute, University College London, London, United Kingdom.
³ UCL Medical School, University College London, London, United Kingdom.
⁴ Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, United Kingdom.
⁵ Department of Histopathology, Royal National Orthopaedic Hospital, London, United Kingdom.

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.