Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases
- 18 April 2016
- journal article
- review article
- Published by Springer Science and Business Media LLC in Nature Reviews Genetics
- Vol. 17 (5), 300-312
- https://doi.org/10.1038/nrg.2016.28
Abstract
Wild-type CRISPR–Cas9 nucleases can induce high-frequency mutations at unintended off-target sites. Methods for defining genome-wide specificities of CRISPR–Cas9 nucleases include cell-based methods such as integrase-defective lentiviral vector (IDLV) capture, genome-wide unbiased identification of DSBs enabled by sequencing (GUIDE-seq), high-throughput genome-wide translocation sequencing (HTGTS), and breaks labelling, enrichment on streptavidin and next-generation sequencing (BLESS), and in vitro methods such as digested genome sequencing (Digenome-seq). Methods for improving genome-wide specificity include truncated guide RNAs (tru-gRNAs), extended gRNAs, paired Cas9 nickases (Cas9n), dimeric RNA-guided FokI–dCas9 ('dead' Cas9) nucleases (RFNs) and engineered variants with reduced non-specific protein–DNA interactions. For many research applications, simple controls (such as the use of multiple gRNAs) and/or genetic reversion or complementation experiments, may help to exclude the possibility of confounding off-target effects. For therapeutic applications, it will be important to carefully define even low-frequency off-target effects using sensitive, unbiased genome-wide methods.Keywords
This publication has 94 references indexed in Scilit:
- Structure and Engineering of Francisella novicida Cas9Cell, 2016
- Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seqGenome Research, 2016
- Generation of mutant mice via the CRISPR/Cas9 system using FokI-dCas9Scientific Reports, 2015
- Rational design of a split-Cas9 enzyme complexProceedings of the National Academy of Sciences, 2015
- Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cellsNature Biotechnology, 2014
- Improving CRISPR-Cas nuclease specificity using truncated guide RNAsNature Biotechnology, 2014
- megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineeringNucleic Acids Research, 2013
- Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing SpecificityCell, 2013
- CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineeringNature Biotechnology, 2013
- Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotesNucleic Acids Research, 2011