Improving CRISPR-Cas nuclease specificity using truncated guide RNAs

By Yanfang Fu, Jeffry D. Sander, Deepak Reyon, Vincent M Cascio, J. Keith Joung. 2014

1,980 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

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Citations in the corpus

CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future (2020, background): “Many features in an sgRNA determine specificity including the seed sequence (a 10–12 bp region proximal to PAM on 3 of spacer sequence) (29, 53), GC content (54, 55), and modifications such as 5 truncation of the sgRNA (56).”
Target specificity of the CRISPR-Cas9 system (2014, positive): “The limitation of this strategy is that nicks induced by Cas9 could still lead to mutations in off target sites via unknown mechanisms [13,29,60,63].”
Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins (2014, background): “Interestingly, truncated sgRNAs can also decrease undesired off-target mutations, improving the specificity of RGENs (Fu et al. 2014).”
Development and Applications of CRISPR-Cas9 for Genome Engineering (2014, positive): “with multiplex nicking to further reduce off-target mutagenesis (Fu et al., 2014).”
Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement (2017, background): “…including selecting highly specific target sequences, optimizing nuclease expression, using truncated sgRNAs, dCas9-FokI fusions, and paired Cas9-nickases (Fujii et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013; Cho et al., 2014; Fu et al., 2014; Guilinger et al., 2014; Tsai et al., 2014).”
Whole organism lineage tracing by combinatorial and cumulative genome editing (2016, background): “Finally, sgRNA sequences 357 and lengths (31), Cas9 cleavage activity and target preferences (32, 33), and the means by which 358 Cas9 and sgRNA(s) are expressed (e.”