2014
DOI: 10.1038/nbt.2916
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Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease

Abstract: RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from… Show more

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Cited by 699 publications
(654 citation statements)
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“…However, CRISPR/Cas9 was found to tolerate MMs between its sgRNA and target sites (Duan et al, 2014;Kuscu et al, 2014;Lin et al, 2014b). We selected the sgRNAs according to the guidelines of increasing the number and position of …”
Section: Discussionmentioning
confidence: 99%
See 1 more Smart Citation
“…However, CRISPR/Cas9 was found to tolerate MMs between its sgRNA and target sites (Duan et al, 2014;Kuscu et al, 2014;Lin et al, 2014b). We selected the sgRNAs according to the guidelines of increasing the number and position of …”
Section: Discussionmentioning
confidence: 99%
“…Based on the pres-ence of a PAM with a 59-NGG sequence, a total of 124 possible sgRNAs can be designed to target the 1059 bp CCR5 ORF or its complementary strand (Table S1, available in the online Supplementary Material). CRISPR/Cas9 was found to tolerate mismatches (MMs) between the sgRNA sequence and recognition site and the properties of MMs, in particular the number and location of MMs, were shown to affect off-target effects (OTEs) Kuscu et al, 2014). Therefore, we further selected sgRNAs among the 20 top-ranked sgRNAs of the 124 candidates by applying the following rules: (1) MMs i2 bp; (2) for MMs52 bp, both locate in the PAM-proximal half; (3) for MMs53 bp, at least 1 bp locates in the PAM-proximal half; and (4) for overlapping sgRNAs with only 1 bp difference, choose the sgRNA with the highest score.…”
Section: Design Of Sgrna Guide Sequencesmentioning
confidence: 99%
“…We also compared cleavage sites identified in this study with those captured by chromatin immunoprecipitation sequencing (ChIP-seq) using catalytically dead Cas9 (dCas9) (Kuscu et al 2014). Strikingly, a vast majority of Cas9-cleaved sites (288 sites, 98%) identified by Digenome-seq were not bound by dCas9 (Supplemental Fig.…”
Section: Digenome-seq Vs Other Methodsmentioning
confidence: 99%
“…In principle, such cis-elements are commonly situated within accessible chromatin domains [1] permissive to dCas9/sgRNA binding [10,11]. It came to our attention that a very recent report identified a novel upstream (~20 kb) enhancer region for human IFNB1, where a bi-directional eRNA transcription was also observed following viral stimulation [12].…”
mentioning
confidence: 99%