Table 1 A summary of methods for identifying genome-wide CRISPR-Cas off-target sites
From: Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing
Categories | Detection methods | Key strengths | Key weaknesses |
|---|---|---|---|
In silico | Bioinformatic prediction based on sequence similarity Cas-OFFinder, CasOT, E-CRISP, COSMID, CROP-IT, CCTOP, Bowtie2, CNN_std, Elevation, FlashFry, predictCRISPR, Crisflash, Synergizing CRISPR, CRISPRitz, MOFF | High throughput | High false positive |
In vitro | Whole-genome sequencing of Cas nuclease-digested DNA/chromatin Digenome-seq, DIG-seq | High sensitivity | Cost-ineffective |
DSB end enrichment by DNA circles digestion CIRCLE-seq, CHANGE-seq | High sensitivity | High skill requirement | |
DSB end enrichment by tagging the cleaved-DNA with Biotin SITE-seq | High sensitivity | Requires in cellulo validation | |
In vitro in cellulo | Labeling of off-target sites by capturing R-loop mediated ssDNA CROss-seq | Unbiased | Narrow time-window |
In cellulo | Indirect labeling of off-target breaks by repaired products GUIDE-seq, iGUIDE, GUIDE-tag HTGTS, LAM-HTGTS, PEM-seq, CAST-seq, UDiTaS IDLV capture, ITR-Seq PolyA-seq PEAC-seq, TAPE-seq | High sensitivity High sensitivity; applicable in vivo Unbiased Unbiased High specificity; applicable in vivo | Require efficient dsODNs delivery into cells Require a large number of input DNA Lower-sensitivity; high false positive Requires efficient LINE-1 delivery into cells Requires high prime editing efficiency |
Indirect labeling of off-target breaks by DNA repair protein MRE11 DISCOVER-seq | Unbiased; applicable in vivo | Narrow time-window | |
Direct labeling of unrepaired break ends in situ BLESS, BLISS | Unbiased | Narrow time-window; high background |
