Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells

By Xuebing Wu, David A. Scott, Andrea J. Kriz, Anthony C Chiu, P. Hsu, Daniela Dadon, A. Cheng, Alexandro E. Trevino, Silvana Konermann, Sidi Chen and 3 more. Nature Biotechnology, 2014

961 citations recorded by the source corpus.

Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.

Subfields: Cardiology and Cardiovascular Medicine, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/a5b90186e6f2cd6a1e4ba14f5f27e7741b17c67e

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Cardiology and Cardiovascular Medicine

In Molecular Biology

Citations in the corpus

Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation (2014, background): “Second, CRISPRi activity is highly sensitive to mismatches between the sgRNA and target DNA, suggesting that off-target binding of dCas9 observed in ChIP-seq experiments is too transient to impact transcription (Duan et al., 2014; Kuscu et al., 2014; Wu et al., 2014).”
In Silico Predictive Modeling of CRISPR/Cas9 guide efficiency (2015, background): “In particular, biochemical and structural studies have shown that particular parts of the 20mer guide participate in step-wise association with DNA 22,23 , suggesting that the thermodynamic properties of different guide segments may yield useful features.”
Target specificity of the CRISPR-Cas9 system (2014, neutral): “On the other hand, at a constant level of Cas9 protein, titrating the amount of sgRNA expression plasmid transfected, and thus the abundance of sgRNA, largely determines the number of off-target binding sites in the mouse genome [37].”
The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells (2016, positive): “In 42–44 This region is necessary for DNA binding, and the seed region and PAM alone are sufficient for specific targeting by Spy Cas9.”
The new frontier of genome engineering with CRISPR-Cas9 (2014, positive): “Results showed that in both human embryonic kidney (HEK293) cells (83) and mouse embryonic stem cells (mESCs) (84), a catalytically inactive version of Cas9 bound to many more sites than those matching the sequence of the sgRNA used in each case.”
CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes (2015, background): “…et al., 2013; Hsu et al., 2014; Hwang et al., 2013; Ikmi et al., 2014; Irion et al., 2014; Jinek et al., 2013; Li et al., 2013a; Li et al., 2013b; Long et al., 2014; Ma et al., 2014; Mali et al., 2013c; Niu et al., 2014; Smith et al., 2014a; Wu et al., 2013; Wu et al., 2014b; Yang et al., 2013).”
Highly Specific Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements (2015, neutral): “Previous reports have found evidence that dCas9 promiscuously binds the genome outside of the target site 29, 30, 41 .”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, background): “Detailed analyses have revealed that the probability of off-target binding events largely depends on chromatin accessibility (89, 90).”