Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

Abstract

CRISPR-Cas gene editing has revolutionized experimental molecular biology over the past decade and holds great promise for the treatment of human genetic diseases. Here we review the development of CRISPR-Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors, focusing on the assessment and improvement of their editing precision and safety, pushing the limit of editing specificity and efficiency. We summarize the capabilities and limitations of each CRISPR tool from DNA editing to RNA editing, and highlight the opportunities for future improvements and applications in basic research, as well as the therapeutic and clinical considerations for their use in patients.

Fig. 1. Overview of CRISPR-Cas nucleases.

a–c CRISPR–Cas9 nucleases and CRISPR-Cas12 nucleases are RNA-guided DNA editing tools, whereas CRISPR-Cas13 nucleases have RNA-guided RNA endonuclease activity. Besides targeted cutting, CRISPR-Cas12/13 nucleases also have collateral cutting activity on non-target DNA/RNA templates. Streptococcus pyogenes (SpCas9) recognizes a relatively common 3’ NGG PAM, and functions optimally with 20-nt spacers, while Cas12a orthologs generally use 5’ T-rich PAMs. PAM protospacer adjacent motif, PFS protospacer flanking site.

Fig. 2. Methods for identifying genome-wide CRISPR-Cas off-target sites.

Genome-wide methods for off-target detection can be divided into two groups: cell-free methods (in vitro) and cell-based methods (in cellulo). Cell-free methods include Digenome-seq (digested genome sequencing) and its improved version DIG-seq that use genomic DNA or chromatin as template during in vitro digestion; CIRCLE-seq (circularization for in vitro reporting of cleavage effects by sequencing) and its improved version CHANGE-seq (circularization for high-throughput analysis of nuclease genome-wide effects by sequencing) that use DNA circles as template; and SITE-seq (selective enrichment and identification of tagged genomic DNA ends by sequencing) that tags the DSBs with biotin-labeled adapter for enrichment. CROss-seq (CRISPR Off-targeting ssDNA sequencing) could be used both in vitro and in cellulo, capturing CRISPR-Cas induced R-loops by N₃-Kethoxal labeling. Cell-based methods include GUIDE-seq (genome-wide, unbiased identification of DSBs evaluated by sequencing) and its improved versions iGUIDE and GUIDE-tag that utilize dsODNs insertions at DSBs in cellulo; HTGTS (high-throughput, genome-wide translocation sequencing), as well as its improved versions LAM-HTGTS and PEM-seq, and similar methods CAST-seq and UDiTaS, can capture translocations between CRISPR-Cas induced on-target (as “bait”) and off-targets (as “preys”); IDLV capture (integrase-deficient lentiviral vector) uses lentiviral vector integrations to identify off-targets, while a similar technique ITR-Seq identifies off-targets by capturing the insertions of specific AAV vector sequences called inverted terminal repeats (ITRs); PolyA-seq detects off-targets by capturing de novo LINE-1 retrotransposon insertions at CRISPR-Cas induced DSBs; PEAC-seq (Prime Editor Assisted off-target Characterization), as well as a similar technique TAPE-seq (TAgmentation of Prime Editor sequencing), tags the on- and off-targets by adopting the prime editor with Cas9 nuclease and a sequence-optimized tag-containing pegRNA; DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing) tracks the precise recruitment of MRE11 at DSBs; and BLESS (direct in situ breaks labeling, enrichment on streptavidin, and next-generation sequencing), as well as its improved version BLISS (breaks labeling in situ and sequencing), directly captures unrepaired DSBs by in situ ligation of biotinylated adapters (for BLESS) or T7 promotor sequence-containing adapters (for BLISS) into off-target breaks.

Fig. 3. Overview of DNA base editors and prime editors.

a Diagram of the DNA base editors. BE2, BE3, HF-BE3, eA3A-BE3, BE4, BE4-Gam, and AncBE4max are developed as cytosine base editors (CBEs), while ABEmax and ABE8e are adenine base editors (ABEs). GBE and CGBE1 belongs to C-to-G base editors (CGBEs). A&C-BEmax, SPACE, Target-ACEmax, STEME-1 and ACBE are generated as dual-deaminase base editors (ACBEs) by fusing CBE with ABE. When fused a CGBE with ABE, AGBEs are developed as represented by AGBE-4 and miniAGBE-4. b Diagram of the Prime editors. While nCas9 (H840A) and pegRNA are required for all prime editing strategies, PE3/PE5/PEmax contains a nicking sgRNA to increase the editing efficiency. PE3b contains a sgRNA with spacer that match the edited strand to minimize the presence of concurrent nicks. Besides one pegRNA-mediated conventional PEs, two pegRNAs strategy is used in dual-pegRNAs, PRIME-Del, HOPE, twinPE, GRAND, Bi-PE and PETI to further increase the prime editing efficiency, enable longer edits and introduction of recombination sites.