Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases

Key Points

  • 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.

Abstract

CRISPR–Cas9 RNA-guided nucleases are a transformative technology for biology, genetics and medicine owing to the simplicity with which they can be programmed to cleave specific DNA target sites in living cells and organisms. However, to translate these powerful molecular tools into safe, effective clinical applications, it is of crucial importance to carefully define and improve their genome-wide specificities. Here, we outline our state-of-the-art understanding of target DNA recognition and cleavage by CRISPR–Cas9 nucleases, methods to determine and improve their specificities, and key considerations for how to evaluate and reduce off-target effects for research and therapeutic applications.

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Figure 1: Targeted methods for defining off-target cleavage effects.
Figure 2: Genome-wide methods for defining off-target cleavage effects.
Figure 3: Methods for improving specificity.

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Acknowledgements

J.K.J. is supported by the US National Institutes of Health (NIH) Director's Pioneer Award (DP1GM105378) and by the Jim and Ann Orr Massachusetts General Hospital (MGH) Research Scholar Award. SQT is supported by an MGH Tosteson Award.

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Correspondence to Shengdar Q. Tsai or J. Keith Joung.

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Competing interests

J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. S.Q.T. and J.K.J. are co-founders of Beacon Genomics, a company that is commercializing methods for determining nuclease specificity.

PowerPoint slides

Glossary

Homology-directed repair

(HDR). A DNA repair pathway that depends on sequence homology to effect repair. A user-supplied 'donor' template can be used to introduce precise alterations of choice with this repair pathway.

Non-homologous end-joining

(NHEJ). A DNA repair pathway in which the double-stranded break (DSB) ends are directly ligated together without a requirement for homology. Variable length insertion or deletion mutations can frequently occur as a consequence of NHEJ-mediated DSB repair.

Point mutations

Genetic changes of a single DNA base pair.

CRISPR

Components of an adaptive immunity system found in bacteria.

CRISPR RNA

(crRNA). Small RNA that contains sequence complementarity to the protospacer and a short repetitive sequence with complementarity to trans-activating crRNA.

Trans-activating crRNA

(tracrRNA). A small trans-encoded RNA that has a portion of sequence complementarity with the CRISPR RNA (crRNA) and is required for Cas9 nuclease activity.

Protospacer

Target sequence for CRISPR interference, flanked by CRISPR repeats.

Protospacer adjacent motif

(PAM). Sequence required to licence Cas9 for cleavage, it is adjacent to the target sequence or protospacer.

Bulges

Gaps in base pairing between target DNA or guide RNA at an RNA-guided nuclease target≈site.

Rolling circle amplification

A method for generating many concatemerized copies of a circular template using a strand-displacing polymerase.

High-throughput sequencing

A method for sequencing populations of DNA molecules, typically with short (<300 bp) reads that have error rates an order of magnitude or more higher than standard long-read Sanger sequencing.

GUIDE-seq

(Genome-wide unbiased identification of DSBs enabled by sequencing). A cell-based method for genome-wide discovery of nuclease-induced double-stranded breaks (DSBs) based on efficient tag integration, tag-specific amplification and high-throughput sequencing.

Double-stranded oligodeoxynucleotide

(dsODN). Used as an integrated genetic tag in genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq).

HTGTS

(High-throughput genome-wide translocation sequencing). A method to detect nuclease-induced off-target double-stranded breaks by observation of translocation junctions.

BLESS

(Breaks labelling, enrichment on streptavidin and next-generation sequencing). A cell-based method for genome-wide discovery of nuclease-induced double-stranded breaks based on cell fixing, nuclei isolation, in situ ligation, enrichment and high-throughput sequencing.

Digested genome sequencing

(Digenome-seq). An in vitro method for detecting Cas9 cleavage of genomic DNA by whole-genome sequencing.

Cas9 nickases

(Cas9n). Engineered variants of Cas9 in which one of the two nuclease domains has been catalytically inactivated, which results in the nicking of only one DNA strand and leaving the other strand intact.

DNA curtains assay

A single-molecule assay for the visualization of protein interactions with individual DNA strands or 'curtains'.

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Tsai, S., Joung, J. Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases. Nat Rev Genet 17, 300–312 (2016). https://doi.org/10.1038/nrg.2016.28

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