. Author manuscript; available in PMC: 2019 May 13.

Published in final edited form as: Nat Protoc. 2018 Nov;13(11):2615–2642. doi: 10.1038/s41596-018-0055-0

TABLE 1|.

Comparison of methods for defining genome-wide CRISPR-Cas9 specificity

Method	Description	Advantages	Limitations
IDLV capture^11,10	Cell-based method where integrase-defective lentiviral vectors are integrated with a selective marker into sites of nuclease-induced DSBs. Vector integration sites are recovered by linear amplification-mediated PCR (LAM-PCR), followed by high-throughput sequencing.	Certain cell types may be more amenable to infection with IDLV than transfection with dsODN tag.	Relatively insensitive due to the integration efficiency and the requirement of positive selection to overcome this; high level of background as IDLVs still retain some capability to randomly integrate into the cellular genome in the absence of nuclease-induced DSBs; IDLVs integrations can occur some distance from the nuclease-induced break so it may be more challenging to map sites.
GUIDE-seq^12,13,10	Based on efficient integration of double stranded oligodeoxynucleotide (dsODN) tags at DSBs by NHEJ in living cells, followed by tag-specific amplification and high-throughput sequencing.	High efficiency of dsODN integration into DSBs enhances sensitivity; quantitative correlation between the numbers of GUIDE-seq reads with mutation frequencies in living cells.	Requires efficient cellular transfection of the dsODN tag, which can be challenging in sensitive cell types or in vivo settings.
HTGTS^15,16,10	Detects off-target nuclease-induced DSBs by observation of translocation junctions between two nuclease-induced DSBs.	Can be applied to discovering nuclease-induced off-targets where the nucleases are delivered in vivo.	Nuclease-induced translocations are rare; translocations occur more frequently with sites in the same chromosome or in close nuclear proximity.
BLESS/BLISS^17,18,10, End-seq²⁰, DSBCapture¹⁹	Based on in situ ligation of adapters to transient nuclease-induced DSBs in fixed cells.	Do not require delivery and incorporation of exogenous DNA for detection	Lack information about nuclease-induced DSBs that were previously repaired by the cell repair machinery.
Digenome-seq^21,22,10	In vitro method based on detection of Cas9-cleaved genomic DNA by whole genome sequencing.	Does not require PCR; has also been tested with base editors.	Does not enrich for nuclease-cleaved sequences and requires a large number of sequencing reads (~400 million); high level of background; yields only one-half of the cleaved site; lacks information about how cellular factors affect nuclease off-target activity.
Site-seq²³	An in vitro method based on Cas9-cleavage of high molecular weight DNA, followed by enzymatic fragmentation, biotinylated adapter ligation, enrichment and sequencing.	Enriches for nuclease-cleaved fragments; reduces sequencing reads required.	Reads contain only one-half of the cleaved sites; lacks information about how cellular factors affects nuclease off-target activity.
CIRCLE-seq⁹	In vitro method where genomic DNA is randomly fragmented, followed by circularization and generation of covalently closed dsDNA molecules. Circular dsDNAs are cleaved by Cas9 at on- and off-target sites, allowing the selective sequencing of nuclease-induced DSBs.	High enrichment so fewer reads required (3-5 million reads); reads contain both halves of the cleavage sites	Lacks information about how cellular factors affects nuclease off-target activity; requires large amount of gDNA.