Use of deactivated Cas9 to facilitate genome editing genes with lethality or welfare concerns.
The high efficiency of CRISPR/Cas9-based genome editing frequently results in bi-allelic mutations when introduced into embryos. Where a mutation is associated with compromised viability, fertility or welfare, the efficiency of animal production can be severely impacted.  To reduce the mutagenic effect of Cas9, we piloted a strategy involving the addition of deactivated Cas9 (dCas9) to produce point mutation mouse models in 11 genes that are reported as homozygous lethal or associated with welfare issues. We showed that combining dCas9 with Cas9 led to an increased recovery of pups, using the 70:30, 80:20 or 90:10 dCas9:Cas9 ratios, and increased preservation of the unedited, wild-type allele using the 80:20 or 90:10 ratios. An 80:20 dCas9:Cas9 ratio increased the overall production rate of mutated founders per manipulated embryo and, importantly, did not hinder germline transmission of the desired allele.  In addition, editing genes in a paralog cluster with a combination of dCas9 and Cas9 reduced unwanted off-target editing, illustrating a further potential applicability of this approach.  This study defines the optimal ratio between dCas9 and Cas9 for strategies aimed at achieving mono-allelic mutations within mosaic founders and proposes a means to reduce the incidence of off-target effects in experiments with limited sgRNA options.
The high efficiency of CRISPR/Cas9-based genome editing frequently results in bi-allelic mutations when introduced into embryos. Where a mutation is associated with compromised viability, fertility or welfare, the efficiency of animal production can be severely impacted.  To reduce the mutagenic effect of Cas9, we piloted a strategy involving the addition of deactivated Cas9 (dCas9) to produce point mutation mouse models in 11 genes that are reported as homozygous lethal or associated with welfare issues. We showed that combining dCas9 with Cas9 led to an increased recovery of pups, using the 70:30, 80:20 or 90:10 dCas9:Cas9 ratios, and increased preservation of the unedited, wild-type allele using the 80:20 or 90:10 ratios. An 80:20 dCas9:Cas9 ratio increased the overall production rate of mutated founders per manipulated embryo and, importantly, did not hinder germline transmission of the desired allele.  In addition, editing genes in a paralog cluster with a combination of dCas9 and Cas9 reduced unwanted off-target editing, illustrating a further potential applicability of this approach.  This study defines the optimal ratio between dCas9 and Cas9 for strategies aimed at achieving mono-allelic mutations within mosaic founders and proposes a means to reduce the incidence of off-target effects in experiments with limited sgRNA options.
Mary Lyon Centre, MRC Harwell Institute