Journal
NUCLEIC ACIDS RESEARCH
Volume 41, Issue 20, Pages 9584-9592Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkt714
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Funding
- National Institutes of Health as an NIH Nano-medicine Development Center Award [PN2EY018244]
- NSF Graduate Research Fellowship [DGE-1148903]
- NIH
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The ability to precisely modify endogenous genes can significantly facilitate biological studies and disease treatment, and the clustered regularly interspaced short palindromic repeats (CRISPR) systems have the potential to be powerful tools for genome engineering. However, the target specificity of CRISPR systems is largely unknown. Here we demonstrate that CRISPR/Cas9 systems targeting the human hemoglobin beta and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin delta and C-C chemokine receptor type 2 genes, respectively, causing gross chromosomal deletions. The guide strands of the CRISPR/Cas9 systems were designed to have a range of mismatches with the sequences of potential off-target sites. Off-target analysis was performed using the T7 endonuclease I mutation detection assay and Sanger sequencing. We found that the repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions and point mutations. Therefore, CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites, including those with mismatches to the 12-bases proximal to the guide strand protospacer-adjacent motif.
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