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...

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Autores principales:	Matthew L. Schwartz, M. Wayne Davis, Matthew S. Rich, Erik M. Jorgensen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Genetics QH426-470
Acceso en línea:	https://doaj.org/article/dcfd7dde31fe417bae680c24232c6f3a
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spelling	oai:doaj.org-article:dcfd7dde31fe417bae680c24232c6f3a2021-11-25T05:53:02ZHigh-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome1553-73901553-7404https://doaj.org/article/dcfd7dde31fe417bae680c24232c6f3a2021-11-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601624/?tool=EBIhttps://doaj.org/toc/1553-7390https://doaj.org/toc/1553-7404Gene 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. Author summary Germlines have evolved specialized mechanisms to protect themselves from invasions by transposons and viruses, which create barriers to genome editing techniques. For example, transgenes are silenced in the germline of the nematode C. elegans, thereby creating a barrier to CRISPR editing by Cas9. To facilitate gene editing, we built a collection of C. elegans strains in which Cas9 is never silenced. CRISPR is significantly more efficient in these animals, decreasing the effort researchers need to expend to get edited animals. The strains are available in multiple genetic backgrounds, and contain accessory transgenes to simplify downstream genetics. Together, these strains enable efficient, low-cost genome editing in C. elegans.Matthew L. SchwartzM. Wayne DavisMatthew S. RichErik M. JorgensenPublic Library of Science (PLoS)articleGeneticsQH426-470ENPLoS Genetics, Vol 17, Iss 11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Genetics QH426-470
spellingShingle	Genetics QH426-470 Matthew L. Schwartz M. Wayne Davis Matthew S. Rich Erik M. Jorgensen High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome
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. Author summary Germlines have evolved specialized mechanisms to protect themselves from invasions by transposons and viruses, which create barriers to genome editing techniques. For example, transgenes are silenced in the germline of the nematode C. elegans, thereby creating a barrier to CRISPR editing by Cas9. To facilitate gene editing, we built a collection of C. elegans strains in which Cas9 is never silenced. CRISPR is significantly more efficient in these animals, decreasing the effort researchers need to expend to get edited animals. The strains are available in multiple genetic backgrounds, and contain accessory transgenes to simplify downstream genetics. Together, these strains enable efficient, low-cost genome editing in C. elegans.
format	article
author	Matthew L. Schwartz M. Wayne Davis Matthew S. Rich Erik M. Jorgensen
author_facet	Matthew L. Schwartz M. Wayne Davis Matthew S. Rich Erik M. Jorgensen
author_sort	Matthew L. Schwartz
title	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_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_sort	high-efficiency crispr gene editing in c. elegans using cas9 integrated into the genome
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/dcfd7dde31fe417bae680c24232c6f3a
work_keys_str_mv	AT matthewlschwartz highefficiencycrisprgeneeditingincelegansusingcas9integratedintothegenome AT mwaynedavis highefficiencycrisprgeneeditingincelegansusingcas9integratedintothegenome AT matthewsrich highefficiencycrisprgeneeditingincelegansusingcas9integratedintothegenome AT erikmjorgensen highefficiencycrisprgeneeditingincelegansusingcas9integratedintothegenome
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High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

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