Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.
basic_science · Level V
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- Record sourced from PubMed, PMID 24463574.
- Also identified by DOI 10.1038/nbt.2808 and PMC identifier 3988262.
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Abstract
Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools. CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended target. Here we report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Our results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.
Medical subject headings
- Bacterial Proteins
- CRISPR-Associated Proteins
- Endonucleases
- RNA, Guide, CRISPR-Cas Systems