High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity

By V. Pattanayak, Steven Lin, J. Guilinger, E. Ma, Jennifer A. Doudna, David R. Liu. Nature Biotechnology, 2013

1,433 citations recorded by the source corpus.

The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 1012 potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/2d836314ed16c6eabe94fcca4c22919083545da6

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In Molecular Biology

Citations in the corpus

MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens (2014, background): “Second, as different sgRNAs targeting the same gene might have different specificities [9-11] and knockout efficiencies, a robust method is needed to take these factors into account in the aggregation of information from multiple sgRNAs (see Figure S2 in Additional file 1 for an example).”
Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA (2014, positive): “This is consistent with previous reports that the 10–12 bp PAM-proximal ‘‘seed’’ region is critical for the Cas9-catalyzed DNA cleavage (Cong et al., 2013; Fu et al., 2013; Hsu et al., 2013; Jinek et al., 2012; Mali et al., 2013a; Pattanayak et al., 2013).”
Loss-of-function approaches in comparative physiology: is there a future for knockdown experiments in the era of genome editing? (2019, background): “However, the majority of off-target sites exhibit mutation frequencies at least 2-fold lower than on-target mutation frequency if the off-target site has greater than one bp mismatch compared with the on-target site (Hsu et al., 2013; Pattanayak et al., 2013; Cho et al., 2014).”
CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes (2015, background): “The specificity of CRISPR/Cas9 is largely dictated by PAM and the 17–20 nt sequence at the 5′ end of gRNAs (Cong et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Mali et al., 2013c; Pattanayak et al., 2013; Wu et al., 2014a).”
The new frontier of genome engineering with CRISPR-Cas9 (2014, neutral): “The amount of Cas9 enzyme expressed in the cell is an important factor in tolerance to mismatches (138).”
Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila (2014, background): “Early studies in human cells did suggest that offtarget mutagenesis also is prevalent for CRISPR/Cas (36, 37).”
Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities (2020, neutral): “It was shown for Cas9 that less than 5% of crRNA sequences with two or more mismatches to the target sequence is functional in target binding (Anderson et al., 2015) and mismatches near the PAM region are less tolerated (Pattanayak et al., 2013).”
Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery (2014, background): “DOI: 10.7554/eLife.04766 4 of 13 Research advance Off-target editing increases with increasing nucleic acid-based delivery of Cas9 (Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013; Mali et al., 2013b).”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “As an example, the PAM from Neisseria meningitidis Cas9 is reported to be 50-NNNNGATT-30 (Jiang et al., 2013; Ma et al., 2014).”
WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system (2015, neutral): “Experimental analysis indicates that Cas9-based genome editing could have wide spread off-target effects, resulting in a significant level of nonspecific editing at other unintended genomic loci [14, 18-20].”