The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double-strand breaks (DSBs) may trigger non-homologous end joining (NHEJ) repair, leading to frameshift mutations, or homology-directed repair (HDR) using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of DSBs. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kilobases to greater than one megabase. We observed a high frequency of intended genomic deletions. Non-deleted alleles are nonetheless often edited with inversions or small indels produced at CRISPR recognition sites. Deleted alleles also typically include small indels at predicted deletion junctions. Authors retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.
0 comments:
Post a Comment