Targeted recombination of homologous chromosomes using CRISPR‐Cas9

Abstract

CRISPR mutagenesis is an efficient way to disrupt specific target genes in many model organisms. We previously devised a targeted CRISPR recombination method to generate intragenic recombinants of alleles in Drosophila. Here, we assessed the applicability of CRISPR targeting‐induced recombination to different genetic loci. We compared the ectopic recombination rates in the male germline by CRISPR targeting at two neighboring genetic loci within the genomic region that consists of the repressed chromatin domain of the Lobe gene, and the transcriptionally active domain of PRAS40. Targeting around the transcription initiation of PRAS40 resulted in higher recombination rates of homologous chromosomes than targeting at the Lobe intron. Based on the efficient homologous recombination by CRISPR targeting observed around transcriptionally active loci, we further investigated targeted recombination between P‐elements that are inserted at different genomic locations. Male recombination by CRISPR targeting of P‐elements located proximally and distally to the ebony gene produced recombinants deficient for the intervening region of ebony transcription. Taken together, we suggest that targeted homologous recombination by CRISPR targeting may have specific genetic applications, such as generation of allelic combinations or chromosomal variations.