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High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome
Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germ...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601624/ https://www.ncbi.nlm.nih.gov/pubmed/34748534 http://dx.doi.org/10.1371/journal.pgen.1009755 |
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author | Schwartz, Matthew L. Davis, M. Wayne Rich, Matthew S. Jorgensen, Erik M. |
author_facet | Schwartz, Matthew L. Davis, M. Wayne Rich, Matthew S. Jorgensen, Erik M. |
author_sort | Schwartz, Matthew L. |
collection | PubMed |
description | Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50–100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits. |
format | Online Article Text |
id | pubmed-8601624 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-86016242021-11-19 High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome Schwartz, Matthew L. Davis, M. Wayne Rich, Matthew S. Jorgensen, Erik M. PLoS Genet Methods Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50–100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits. Public Library of Science 2021-11-08 /pmc/articles/PMC8601624/ /pubmed/34748534 http://dx.doi.org/10.1371/journal.pgen.1009755 Text en © 2021 Schwartz et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Methods Schwartz, Matthew L. Davis, M. Wayne Rich, Matthew S. Jorgensen, Erik M. High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome |
title | High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome |
title_full | High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome |
title_fullStr | High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome |
title_full_unstemmed | High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome |
title_short | High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome |
title_sort | high-efficiency crispr gene editing in c. elegans using cas9 integrated into the genome |
topic | Methods |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601624/ https://www.ncbi.nlm.nih.gov/pubmed/34748534 http://dx.doi.org/10.1371/journal.pgen.1009755 |
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