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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2021-11-01
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| Series: | PLoS Genetics |
| Online Access: | https://doi.org/10.1371/journal.pgen.1009755 |
| Summary: | 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. |
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| ISSN: | 1553-7390 1553-7404 |