Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Schwartz, Matthew L., Davis, M. Wayne, Rich, Matthew S., Jorgensen, Erik M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
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
_version_ 1784601395008110592
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
work_keys_str_mv AT schwartzmatthewl highefficiencycrisprgeneeditingincelegansusingcas9integratedintothegenome
AT davismwayne highefficiencycrisprgeneeditingincelegansusingcas9integratedintothegenome
AT richmatthews highefficiencycrisprgeneeditingincelegansusingcas9integratedintothegenome
AT jorgensenerikm highefficiencycrisprgeneeditingincelegansusingcas9integratedintothegenome