Article Synopsis
- CRISPR RNA-guided nucleases (RGNs) like Cas9 are powerful tools for genome editing but can unintentionally cause mutations at similar DNA sites.
- Truncated guide RNAs (gRNAs) with less than 20 complementary nucleotides can significantly reduce these undesired mutations by up to 5,000 times without compromising the accuracy of targeted edits.
- This research presents a straightforward method to enhance the specificity of Cas9 nucleases and paired nickases, ultimately improving genome editing precision.
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Article 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.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988262 | PMC |
| http://dx.doi.org/10.1038/nbt.2808 | DOI Listing |
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