Table 2.
Important features that influence the utility of off-target detection methods.
| Method | References | Category | Sensitivity * | Input DNA | Detection state | Detection mechanism | Edits detected | Genomic context for nuclease | Target enrichment |
|---|---|---|---|---|---|---|---|---|---|
| Digenome-seq | Kim et al., 2015 | In vitro | 0.1% | 1 μg | Target-site DSB | Sequence alignment pile-up | DSBs | Cell free DNA | None |
| DIG-Seq | Kim and Kim, 2018 | In vitro | 0.1% | 1 μg | Target-site DSB | Sequence alignment pile-up | DSBs | Chromatin environment | None |
| SITE-Seq | Cameron et al., 2017 | In vitro | 0.1% | 7.5 μg | Target-site DSB | Sequence alignment pile-up | DSBs | Cell free DNA | Streptavidin selection |
| CIRCLE-seq | Tsai et al., 2017 | In vitro | 0.01% | 25 μg | Target-site DSB | In vitro cleavage | DSBs | In vitro genomic library | Linearization of circularized library |
| CHANGE-seq | Lazzarotto et al., 2020 | In vitro | NR** | 5 μg | Target-site DSB | In vitro cleavage | DSBs | In vitro genomic library | Linearization of circularized library |
| VIVO | Akcakaya et al., 2018 | In vivo | 0.13% | 25 μg | Target-site mutation | Targeted sequencing | Repair site mutations (indels) | In organism | Targeted sequencing |
| GUIDE-seq | Tsai et al., 2015 | Ex vivo | 0.12% | 800 ng | Repaired with oligonucleotide incorporation | Oligonucleotide uptake by NHEJ | NHEJ repair sites | Cellular environment | Anchored primer amplification |
| UDiTaS | Giannoukos et al., 2018 | Ex vivo | 0.1% (all edits), 0.01% (translocations) | 50 ng | Repaired with large deletions, inversions, translocations | Targeted sequencing | Repair site mutations (indels), translocations, inversions, large deletions | Cellular environment | Anchored primer amplification |
| LAM-PCR HTGTS | Frock et al., 2015; Hu et al., 2016 | Ex vivo | NR** | 20–100 μg | Translocation junction | Translocation bait and prey | Translocations | Cellular environment | Anchored primer amplification |
| DISCOVER-seq | Wienert et al., 2019, 2020 | In vivo | 0.3% | 2 × 106 – 1 × 107 cells; 40–80 mg homogenized tissue | Unrepaired DSB during DNA damage response (DDR) | MRE11 antibody labeling | Unrepaired DSBs | In organism | ChIP |
| BLISS | Yan et al., 2017 | In situ | NR** | 100 μL of nuclei suspension from cells on 13 mm coverslips | Unrepaired DSB in situ | In situ end-capture | Unrepaired DSBs | Cellular environment | Transcription |
*
Sensitivity is defined as the frequency of occurrence on a per cell basis in a cell population. For example a sensitivity of 0.1% refers to an editing event which occurs in 1 out of 1,000 cells.
**
Not reported (NR) in the cited manuscript.