Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity
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 und...
| Main Authors: | , , , , , , , , , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
Cell Press/Elsevier
2016
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| Online Access: | http://hdl.handle.net/1721.1/102782 https://orcid.org/0000-0003-2782-2509 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0002-2639-9879 |
| Summary: | 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. |
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