Table 4 |.

Comparison of experimental off-target site search tools with estimated performance characteristics

Name	Method	What is detected?	Setting	Cut site enrichment	Cellular false positives
In vitro
Digenome-seq47	NGS fragment end statistics	DSB	DNA	170*	65%
CIRCLE-seq49	DSB end enrichment	DSB	DNA	821	88%
SITE-seq48	DSB end enrichment	DSB	DNA	178	95%
DIG-seq51	NGS fragment end statistics	DSB	Chromatin	289*	64%
In vivo
HTGTS26	Rearrangement detection	Repair product	Cells	4,700	n.d.
GUIDE-seq27	Oligonucleotide integration	Repair product	Cells	29,000	20%
DISCOVER-seq55	DSB end enrichment	DSB	Cells/tissue	233	∼0%
BLISS54	DSB end tagging	DSB	Cells/tissue	160	20%

Summary of experimental techniques for off-target cutting by programmable nucleases. Because all methods rely on specific enrichment of either DNA close to cut sites or DNA ends close to cut sites, we use the degree to which they achieve enrichment of DNA fragments near the on-target editing site (‘Cut site enrichment’) as a proxy for their sensitivity. For ‘Cellular false positives’, we use the fraction of sites identified by each technique that fail to be edited (indels <0.1%) in the accompanying cellular validation studies; see main text for details.

^*Cut site enrichment for Digenome-seq and DIG-seq was assessed by enrichment of sequencing fragments whose ends are precisely at the on-target editing site.

n.d., not determined.