CRISPR-Cas9 enables conditional mutagenesis of challenging loci

Abstract

The International Knockout Mouse Consortium (IKMC) has produced a genome-wide collection of 15,000 isogenic targeting vectors for conditional mutagenesis in C57BL/6N mice. Although most of the vectors have been used successfully in murine embryonic stem (ES) cells, there remain a set of nearly two thousand genes that have failed to target even after several attempts. Recent attention has turned to the use of new genome editing technology for the generation of mutant alleles in mice. Here, we demonstrate how Cas9-assisted targeting can be combined with the IKMC targeting vector resource to generate conditional alleles in genes that have previously eluded targeting using conventional methods.

Figure 1. CRISPR-assisted conditional mutagenesis strategy.

Single guide RNAs (sgRNAs) are targeted to a small deleted region near the LoxP site in the genomic intron which is absent in IKMC conditional targeting vectors (Δ), resulting in cleavage of the genomic locus but not the targeting vector. Using the dual-nickase strategy, Cas9[D10A] generates a single strand break on both antiparallel strands, triggering repair by homologous recombination. Traditional IKMC long arm homology conditional vectors as well as short arm vectors derived from IKMC intermediate vectors are amenable for assisted mutagenesis. The final allele is indistinguishable from conventional targeted conditional alleles from IKMC with a floxed critical exon that can be removed with Cre recombinase and results in a null allele. The yellow region is deleted in IKMC targeting vectors (Δ). Mock exons are shown as green boxes.