Box 1: Cas9 target recognition, binding and cleavage
Streptococcus pyogenes Cas9 nuclease has been engineered to require only two components: the Cas9 protein and a short ~100 nucleotide guide RNA (gRNA) that form a complex that can recognize and cleave a 20 bp double-stranded DNA target site (known as protospacer DNA) that is complementary to the 5′ end of the gRNA and is next to a protospacer adjacent motif (PAM) of 5′-NGG-3′, in which N can be any nucleotide (see the figure, part a). The single gRNA transcript is an engineered fusion of naturally occurring CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA)17. The tracrRNA was originally discovered by differential RNA sequencing and found to be an essential component for CRISPR interference in S. pyogenes bacteria92. The target specificity of Cas9 is mediated by nucleic acid interactions between the 20 nucleotides at the 5′ end of the gRNA and the protospacer DNA, as well as by protein–DNA interactions between Cas9 protein and the PAM. Upon recognition of a PAM sequence, Cas9 initiates sequential unwinding of the protospacer target-site duplex, stabilized by the formation of an R-loop structure between the protospacer DNA and the gRNA82. Sufficient RNA–DNA complementarity between the gRNA and the target DNA strand triggers a conformational change in Cas9 that activates concerted cleavage of the target DNA strand by the HNH nuclease domain, and of the non-target strand by its RuvC domain87. In vitro, Cas9 nucleases can produce either blunt or 1 bp 5′-staggered ends17, 71. In mammalian cells, Cas9 nuclease-induced double-stranded breaks (DSBs) can be repaired by one of two competing DNA repair pathways. First is the error-prone non-homologous end-joining (NHEJ), which results in insertions or deletions (indels) that are often exploited to create frame-shift or knockout mutations; and second is the precise homology-directed repair (HDR), which in the presence of a user-supplied donor template is often used for gene correction or knock-in experiments (see the figure, part b).
The results of several studies strongly suggest that the cleavage specificity of Cas9 nuclease differs from the binding site specificity of catalytically inactive 'dead' Cas9 (dCas9). For example, chromatin immunoprecipitation followed by sequencing (ChIP–seq) has been used to identify DNA sites bound genome-wide by dCas9 in human and mouse cells. Analysis of off-target binding sites detected by ChIP–seq have shown that very few of these are cleaved or mutagenized by catalytically active Cas9 (Refs 93,94,95), consistent with the proposed mechanism that more extensive pairing of the gRNA mediates a conformational change that enables Cas9 cleavage93, 94. This conformational gating mechanism may explain, in part, why the extent of protospacer complementarity that is required is different for efficient cleavage by wild-type Cas9 nuclease (≥17 bp) versus transcriptional activation by dCas9–activator fusion proteins (≥14 bp)89, 90.
Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129, USA; and the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
- Shengdar Q. Tsai &
- J. Keith Joung
Competing interests statement
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.
Shengdar Q. Tsai
Shengdar Q. Tsai is an instructor at Massachusetts General Hospital, Charlestown, Massachusetts, USA, and Harvard Medical School, Boston, Massachusetts, USA. He has focused on developing methods for high-throughput genome editing with transcription activator-like effector nucleases (TALENs), and defining and improving the genome-wide specificity of CRISPR–Cas9 nucleases. His long-term goal is to develop safe and highly specific targeted genome-editing strategies for treating human genetic conditions.
J. Keith Joung
J. Keith Joung is a Jim and Ann Orr Massachusetts General Hospital (MGH) Research Scholar, pathologist, and Associate Chief of Pathology for Research at MGH, Charlestown, USA, and Professor of Pathology at Harvard Medical School, Boston, Massachusetts, USA. His laboratory develops genome editing technologies that use engineered zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR–Cas9 nucleases. J. Keith Joung's homepage
- 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.
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.
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.
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.
(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).
(High-throughput genome-wide translocation sequencing). A method to detect nuclease-induced off-target double-stranded breaks by observation of translocation junctions.
(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'.