Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/cas9 nuclease system in mammalian cells

Matthew C. Canver, Daniel E. Bauer, Abhishek Dass, Yvette Y. Yien, Jacky Chung, Takeshi Masuda, Takahiro Maeda, Barry H. Paw, Stuart H. Orkin

Research output: Contribution to journalArticle

174 Citations (Scopus)

Abstract

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. 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 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We 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.

Original languageEnglish
Pages (from-to)21312-21324
Number of pages13
JournalJournal of Biological Chemistry
Volume289
Issue number31
DOIs
Publication statusPublished - Aug 1 2014
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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