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

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.

Figure 1 |. Targeted methods for defining off-target cleavage effects.

a An early method for identifying off-target effects was computational prediction off off-target sites followed by targeted analysis by mismatch cleavage assay like T7E1 or high-throughput sequencing. The limitation of these approaches are that they are biased by the assumptions made by computational predictions about the sequence features at off-target sites: to narrow the scope of the sites examined, only in silico-predicted sites are interrogated, thus leaving additional genuine off-target sites undetected. b In vitro site selection of partially randomized libraries. Concatameric libraries are generated by rolling circle amplification of circularized oligonucleotide templates. Cleavage of libraries results in members with newly available ends compatible for ligation with adapters (blue and red) for high-throughput sequencing. Part b is adapted from reference.

Figure 2 |. Genome-wide methods for defining off-target cleavage effects.

a | IDLV capture. Integration defective lentiviruses (IDLVs; green) are integrated with a selectable marker into sites of nuclease-induced double-stranded breaks (DSBs) in living cells. Integration sites are recovered by ligation-adapter-mediated LAM-PCR, followed by high-throughput sequencing^,. b | Genome-wide unbiased identification of DSBs enabled by sequencing (GUIDE-seq). An end-protected short double-stranded oligodeoxynucleotide (dsODN) is efficiently integrated into sites of nuclease-induced DSBs in living cells. This short sequence is used for tag-specific amplification followed by high-throughput sequencing to identify off-target cleavage sites. c | High-throughput genome-wide translocation sequencing (HTGTS). Two nucleases are expressed in a cell, to generate a ‘prey’ and ‘bait’ DSB. Using a biotinylated primer designed against the bait DSB junction, translocations between ‘prey’ and ‘bait’ are recovered by LAM-PCR for high-throughput equencing. Off-target cleavage sites are identified by analysis of these translocation junctions. d | Breaks labeling, enrichment on streptavidin and next-generation sequencing (BLESS^,). Nuclease-treated cells are fixed, intact nuclei are isolated, permeabilized, and sequencing adapters are ligated in situ to transient nuclease-induced DSBs. Adapter-ligated fragments are enriched and amplified for high-throughput sequencing. Part d is adapted from reference. e | Digenome-seq. Genomic DNA is isolated from cells and treated with Cas9 nuclease in vitro. Sequencing adapters are ligated and high-throughput sequencing is performed at standard whole-genome sequencing coverage.

Figure 3 |. Methods for improving specificity.

a | Truncated guide RNAs (tru-gRNAs). Cas9 is directed by gRNAs that are truncated by 2–3 nucleotides on the 5 end. b | gRNA extensions. Cas9 is directed by a gRNA with 2 additional G nucleotides appended to the 5 end. c | Paired nickases. One of the 2 nuclease domains of Cas9 is catalytically inactivated to make an enzymatically active nickase. Co-localization of a pair of nickases oriented in a ‘PAM-out’ orientation, with each nickase independently nicking one strand, results in efficient DSBs^,. Part c is adapted from reference. d | Dimeric RNA-guided FokI-dCas9 nucleases (RFNs). Catalytically inactivated Cas9 (dCas9) is fused to dimerization-dependent FokI non-specific nuclease domain. A pair of FokI-dCas9 monomers oriented in a ‘PAM-out’ orientation mediates efficient DSBs^,. Part d is adapted from reference. e | Engineered Cas9 variants. Cas9 variants are engineered with reduced non-specific DNA interations with the target (SpCas9-HF1) or non-target (eSpCas9 1.1) strands. For further details see the main text and Table 1.