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Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants

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. Stu...

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Autores principales: Usher, Inga, Ligammari, Lorena, Ahrabi, Sara, Hepburn, Emily, Connolly, Calum, Bond, Gareth L., Flanagan, Adrienne M., Cottone, Lucia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9294353/
https://www.ncbi.nlm.nih.gov/pubmed/35865001
http://dx.doi.org/10.3389/fgeed.2022.932434
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author Usher, Inga
Ligammari, Lorena
Ahrabi, Sara
Hepburn, Emily
Connolly, Calum
Bond, Gareth L.
Flanagan, Adrienne M.
Cottone, Lucia
author_facet Usher, Inga
Ligammari, Lorena
Ahrabi, Sara
Hepburn, Emily
Connolly, Calum
Bond, Gareth L.
Flanagan, Adrienne M.
Cottone, Lucia
author_sort Usher, Inga
collection PubMed
description 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.
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spelling pubmed-92943532022-07-20 Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants Usher, Inga Ligammari, Lorena Ahrabi, Sara Hepburn, Emily Connolly, Calum Bond, Gareth L. Flanagan, Adrienne M. Cottone, Lucia Front Genome Ed Genome Editing 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. Frontiers Media S.A. 2022-07-05 /pmc/articles/PMC9294353/ /pubmed/35865001 http://dx.doi.org/10.3389/fgeed.2022.932434 Text en Copyright © 2022 Usher, Ligammari, Ahrabi, Hepburn, Connolly, Bond, Flanagan and Cottone. https://creativecommons.org/licenses/by/4.0/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.
spellingShingle Genome Editing
Usher, Inga
Ligammari, Lorena
Ahrabi, Sara
Hepburn, Emily
Connolly, Calum
Bond, Gareth L.
Flanagan, Adrienne M.
Cottone, Lucia
Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants
title Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants
title_full Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants
title_fullStr Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants
title_full_unstemmed Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants
title_short Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants
title_sort optimizing crispr/cas9 editing of repetitive single nucleotide variants
topic Genome Editing
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9294353/
https://www.ncbi.nlm.nih.gov/pubmed/35865001
http://dx.doi.org/10.3389/fgeed.2022.932434
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