Usher, Inga;
Ligammari, Lorena;
Ahrabi, Sara;
Hepburn, Emily;
Connolly, Calum;
Bond, Gareth L;
Flanagan, Adrienne M;
(2022)
Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants.
Frontiers in Genome Editing
, 4
, Article 932434. 10.3389/fgeed.2022.932434.
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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.
| Type: | Article |
|---|---|
| Title: | Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants |
| Location: | Switzerland |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.3389/fgeed.2022.932434 |
| Publisher version: | https://doi.org/10.3389/fgeed.2022.932434 |
| Language: | English |
| Additional information: | Copyright © 2022 Usher, Ligammari, Ahrabi, Hepburn, Connolly, Bond, Flanagan and Cottone. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
| Keywords: | CRISPR, CRISPR/Cas9, cell line, genome editing, homology directed repair (HDR), prime editing, stem cells |
| UCL classification: | UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Pathology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10153990 |
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