Cas9-induced nonhomologous recombination in C. elegans
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Abstract
Identification of the genetic basis of phenotypic variation within species remains challenging. In species with low recombination rates, such as Caenorhabditis elegans , genomic regions linked to a phenotype of interest by genetic mapping studies are often large, making it difficult to identify the specific genes and DNA sequence variants that underlie phenotypic differences. Here, we introduce a method that enables researchers to induce targeted recombination in C. elegans with Cas9. We demonstrate that high rates of targeted recombination can be induced by Cas9 in a genomic region in which naturally occurring recombination events are exceedingly rare. We anticipate that Cas9-induced nonhomologous recombination (CINR) will greatly facilitate high-resolution genetic mapping in this species.
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Genotyping of the F3 progeny revealed high rates of recombination within the introgressed region for each of the three guides (gSZ71: 55.9% recombinants [19/35]; gSZ73 52.4% recombinants [33/63]; gSZ74: 10% recombinants [2/20]).
This is s great proof-of-concept example that Cas9-mediated nonhomologous recombination can be a platform in C. elegans to aid in genetically mapping variants.
Numerous studies in C. elegans using co-conversion have achieved a similar efficiency of imprecise repair at a Cas9-induced DSB as you achieved for isolating LOH offspring, which is remarkable. Do you have a sense of the cleavage and repair efficiency of Cas9-induced DSBs you've generated-not just LOH animals?
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Given the high rates of Cas9-induced recombination we observed by PCR in a genomic region with undetectable natural recombination
For comparison, from other studies, do you have a sense of the natural recombination efficiency of neighboring loci in this region of chromosome V?
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In this study, we show that Cas9-induced double-stranded DNA breaks facilitate targeted nonhomologous recombination in C. elegans
It would satisfying to see this pattern of repair generalize across different regions of different chromosomes. There may be positional effects that influence the rate of Cas9-mediated recombination.
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Because of the relative ease of generating targeted recombination events at a high frequency, we anticipate CINR will greatly expedite genetic fine-mapping in C. elegans in regions with low recombination rates, including chromosome centers (Rockman & Kruglyak, 2009) and divergent regions (Lee et al., 2021).
It would interesting to examine if one can recover non-homologous recombination events at a reasonable frequency when targeting highly repetitive regions in the genome that may be prone to favor microhomology-mediated repair from sequences in cis.
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Because of the relative ease of generating targeted recombination events at a high frequency, we anticipate CINR will greatly expedite genetic fine-mapping in C. elegans in regions with low recombination rates, including chromosome centers (Rockman & Kruglyak, 2009) and divergent regions (Lee et al., 2021).
It would interesting to examine if one can recover non-homologous recombination events at a reasonable frequency when targeting highly repetitive regions in the genome that may be prone to favor microhomology-mediated repair from sequences in cis.
-
In this study, we show that Cas9-induced double-stranded DNA breaks facilitate targeted nonhomologous recombination in C. elegans
It would satisfying to see this pattern of repair generalize across different regions of different chromosomes. There may be positional effects that influence the rate of Cas9-mediated recombination.
-
Given the high rates of Cas9-induced recombination we observed by PCR in a genomic region with undetectable natural recombination
For comparison, from other studies, do you have a sense of the natural recombination efficiency of neighboring loci in this region of chromosome V?
-
Genotyping of the F3 progeny revealed high rates of recombination within the introgressed region for each of the three guides (gSZ71: 55.9% recombinants [19/35]; gSZ73 52.4% recombinants [33/63]; gSZ74: 10% recombinants [2/20]).
This is s great proof-of-concept example that Cas9-mediated nonhomologous recombination can be a platform in C. elegans to aid in genetically mapping variants.
Numerous studies in C. elegans using co-conversion have achieved a similar efficiency of imprecise repair at a Cas9-induced DSB as you achieved for isolating LOH offspring, which is remarkable. Do you have a sense of the cleavage and repair efficiency of Cas9-induced DSBs you've generated-not just LOH animals?
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