Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity

Details

• Alternative Title:
  Cell
• Personal Author:
  Ran, F. Ann ; Hsu, Patrick D. ; Lin, Chie-Yu ; Gootenberg, Jonathan S. ; Konermann, Silvana ; Trevino, Alexandro ; Scott, David A. ; Inoue, Azusa ; Matoba, Shogo ; Zhang, Yi ; Zhang, Feng
• Description:
  Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.
• Subjects:
  Animals Article Base Sequence DNA Breaks, Double-Stranded Gene Targeting Genome Mice Molecular Sequence Data RNA, Guide Streptococcus Pyogenes Zygote

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• Source:
  Cell. 2013; 154(6):1380-1389.
• Pubmed ID:
  23992846
• Pubmed Central ID:
  PMC3856256
• Document Type:
  Journal Article
• Funding:
  1DP1-MH100706/DP/NCCDPHP CDC HHS/United States ; 1R01-DK097768/DK/NIDDK NIH HHS/United States ; DP1 MH100706/MH/NIMH NIH HHS/United States ; GM68804/GM/NIGMS NIH HHS/United States ; R01 DK097768/DK/NIDDK NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; T32GM007753/GM/NIGMS NIH HHS/United States ; U01DK089565/DK/NIDDK NIH HHS/United States ; Howard Hughes Medical Institute/United States
• Volume:
  154
• Issue:
  6
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:be6087c53535211cb83f21eafef653119f438d0588178c869db1bea34bffa42f
• Download URL:
  https://stacks.cdc.gov/view/cdc/29797/cdc_29797_DS1.pdf
• File Type:
  [PDF - 1.36 MB ]