Table 1.
Categorized sgRNA specificity
| Study | Off-target prediction/ detection method |
Validation rate a |
sgRNA specificity based on validated off-target activity |
|||
|---|---|---|---|---|---|---|
| Highly specific (0 off-targets) |
Intermediate (1–5 off-targets) |
Promiscuous (>5 off-targets) |
||||
| Computational prediction & In vitro screen | Mali et al. 2013 | Reporter gene screen | <1% | n.d.b (artificial target sites) | ||
| Cho et al. 2013 | Computational & Exome capture | <1% | C4BPB CCR5 |
|||
| Fu et al. 2013 | Computational | <1% | RNF2 FANCF |
EMX1 VEGFA site 1 |
VEGFA site 2 (12) VEGFA site 3 (7) |
|
| Hsu et al. 2013 | Computational | <1% | EMX site 1 EMX site 3 |
|||
| Pattanayak et al. 2013 | Computational & In vitro screen | <1% | CLTA4 (3) | |||
| Cradick et al. 2013 | Computational | <1% | 9 sgRNAs c | 10 sgRNAs c | ||
| Lin et al. 2014 | Computational (gRNA and DNA bulges) | <1% | n.d.b (focus on DNA/RNA bulges, not mismatches) | |||
| Genome-wide detection of dCas9 binding | Wu et al. 2014 | ChIP-seq | <1% d | Nonog-sg3 Phc1-sg1 Phc1-sg2 |
Nanog-sg2 | |
| Cecnic et al. 2014 | ChIP-seq | <1% d | sgp53-3 | sgp53-1 | ||
| Kuscu et al. 2014 | ChIP-seq | <1% d | ||||
| O'Geen et al. 2015 | ChIP-seq & Sequence capture | <1% d | S1 | S2 | ||
| Gersbach et al. 2015 | ChIP-seq & RNA-seq | 3–17 % | IL1RN HBG1/2 |
|||
| Genome-wide detection Of DSBs and NHEJ | Tsai et al. 2015 | GUIDE-seq | 80% | RNF2 | HEK 293 site 2 HEK 293 site 3 |
VEGFA site 1 (21) VEGFA site 2 (151) VEGFA site 3 (59) EMX1 (15) FANCF (8) HEK293 site 1 (9) HEK 293 site 4 (133) |
| Kim et al. 2015 | Digenome-seq | 7–10% | HBB | VEGFA (81) | ||
| Ran et al. 2015 | BLESS | 14–41% | Pcsk9 | EMX1-sg1 | EMX-sg2 (12) | |
| Frock et al. 2015 | HTGTS (focus on translocation) | n.d.b | RAG1B | RAG1A | ||
| Wang et al. 2015 | IDLV | 100% e | WAS CR-3 TAT CR-4 TAT CR-6 |
WAS CR-5 TAT CR-1 |
WAS CR-4 (12) | |
a
Validation rate serves only as a reference and is highly dependent on the sensitivity of the method, the number of sgRNAs tested, and the number of potential off-target sites used for validation.
b
n.d., not determined
c
Only one potential off-target site was examined.
d
ChIP-seq detection is based on DNA binding while validation is based on DNA cleavage activity. Both methods have different specificity determinants and cannot be directly compared.
e
Additional sites that were not identified by IDLV were validated after computational prediction.