DNA targeting specificity of RNA-guided Cas9 nucleases

By P. Hsu, David A. Scott, Joshua A. Weinstein, F. Ran, Silvana Konermann, Vineeta Agarwala, Yinqing Li, Eli J. Fine, Xuebing Wu, Ophir Shalem and 4 more. 2013

4,640 citations recorded by the source corpus.

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

Subfields: Molecular Biology

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Citations in the corpus

Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells (2014, positive): “Using our previously validated model 9 , the background indel frequencies due to sequencing noise were determined for each individual target using two biological replicates transfected with only Cas9 but no sgRNA (“control”).”
Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Interactions (2015, methodology): “Heterozygous DelB/+ mice showed shortening of the second and third digits due to hypoplastic phalanges, which was most pronounced in the middle phalanx of the second digit (Figure S2).”
The CRISPR/Cas9 system for plant genome editing and beyond. (2015, background): “Unlike ZFNs and TALENs, the CRISPR/Cas9 system can cleave methylated DNA in human cells (Hsu et al., 2013), allowing genomic modifications that are beyond the reach of the other nucleases (Ding et al.”
CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing (2014, methodology): “CHOPCHOP has a number of features that separate it from the other CRISPR/Cas9 or TALEN tools currently available (Supplementary Table S1) (25,28-34).”
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).”
Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA (2014, background): “…(on-target effect) and specificity (off-target effect) are strongly affected by Cas9 protein and guide RNA concentrations (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013), and (2) it is challenging to exactly control these concentrations so that they are the same in the…”
Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens (2017, methodology): “…medium-scale experiments have been used to guide gRNA selection algorithms (Doench et al. 2016; Haeussler et al. 2016; Heigwer et al. 2014, 2016; Hsu et al. 2013; Liu et al. 2016; Park et al. 2016), to our knowledge our design is based on the largest available set of empirical screening results.”
Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins (2014, background): “Reducing off-target effects of RGENs via RNP delivery RGENs can induce off-target mutations at sites that are highly homologous to on-target sites (Cradick et al. 2013; Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013; Cho et al. 2014).”
Evaluation and Design of Genome-wide CRISPR/Cas9 Knockout Screens (2017, positive): “Though a variety of small and medium scale experiments have been used to guide gRNA selection algorithms [17-23], our design is, to our knowledge, based on the largest available set of empirical screening results.”