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.