Table 3. Comparison of off target mutation in both mouse cell lines and mice induced by tru-RGNs and std-RGNs.
Target sites | Recognition sites |
Mutation frequencies in cells (%) |
Mutant mice / total mice tested (%) |
|||
---|---|---|---|---|---|---|
std-RGNs (20 nt) | Tru-RGNs (17/18 nt) | Std-gRNA Mean ± SEM | Tru-gRNA Mean ± SEM | Std-gRNA Mean ± SEM | Tru-gRNA Mean ± SEM | |
FVII site 1 (on-target) | AAGGCGTGCCAACTCACTCC TGG | GGCGTGCCAACTCACTCC TGG | ||||
OT1–1 | AGGATGGGCCAACTCACTCC TGG | GATGGGCCAACTCACTCC TGG | 13.3 ± 0.8a | 13.1 ± 0.3a | 0a | 0a |
OT1–2 | AGGATGGGCCAACTCACTCC TGG | GATGGGCCAACTCACTCC TGG | 6.6 ± 0.2a | 6.2 ± 0.3a | 0a | 0a |
OT1–3 | AAGCCCTCCTAACTCACTCC CAG | GCCCTCCTAACTCACTCC CAG | 0a | 0a | 0a | 0a |
OT1–4 | AAGCCATCCAAACTCACTCC TGG | GCCATCCAAACTCACTCC TGG | 21.4 ± 0.6a | 20.3 ± 1.0a | 0a | 0a |
OT1–5 | ACTGTGTGCCACCTCACTCC TGG | TGTGTGCCACCTCACTCC TGG | 2.1 ± 0.4a | 0b | 0a | 0a |
FVII site 2 (on-target) | GCGTGCCAACTCACTCCTGG AGG | GTGCCAACTCACTCCTGG AGG | ||||
OT2–1 | GCTGGCAAACTCACTCCTGG AAG | TGGCAAACTCACTCCTGG AAG | 4.4 ± 0.4a | 3.0 ± 0.1b | 0a | 0a |
OT2–2 | GGCTGCCACCTCACTCCTGG AGG | CTGCCACCTCACTCCTGG AGG | 0a | 0a | 0a | 0a |
OT2–3 | GTGTGTCTACTCACTCCTGG GGG | GTGTCTACTCACTCCTGG GGG | 0a | 0a | 0a | 0a |
OT2–4 | CTGTCCCAATTCACTCCTGG TAG | GTCCCAATTCACTCCTGG TAG | 0a | 0a | 0a | 0a |
OT2–5 | AGGTACCAGCTCACTCCTGG GGG | GTACCAGCTCACTCCTGG GGG | 8.5 ± 0.5a | 6.0 ± 0.3b | 0a | 0a |
FVII site 3 (on-target) | GGAGCTTTGGCCCGGCTCTC TGG | GCTTTGGCCCGGCTCTC TGG | ||||
OT3–1 | GGGGCTTTGGCCAGGCTCTC AGG | GCTTTGGCCAGGCTCTC AGG | 0a | 0a | 0a | 0a |
OT3–2 | AGGGCCTTGGCCCGGCTCTC GGG | GCCTTGGCCCGGCTCTC GGG | 3.7 ± 0.5a | 22.0 ± 0.6b | 0a | 29.3 ± 7.1b |
OT3–3 | TAAACTTTGACCCGGCTCTC TAG | ACTTTGACCCGGCTCTC TAG | 5.7 ± 0.4a | 5.2 ± 0.4a | 0a | 0a |
OT3–4 | ACAGCTTTGGCCAGGCTCTC GAG | GCTTTGGCCAGGCTCTC GAG | 6.2 ± 0.3a | 3.2 ± 0.2b | 0a | 0a |
OT3–5 | ACAGCTTTGGCCAGGCTCTC GAG | GCTTTGGCCAGGCTCTC GAG | 7.2 ± 0.1a | 5.6 ± 0.2 | 0a | 0a |
a,b values with the different numbers within the same row showed significant differences (P < 0.05). The frequencies of mutation at predicated off-target (OT) sites were compared separately in either cell lines or newborn mice between each tru-gRNA and std-gRNA group. At each OT site, the different nucleotides mismatch to the targeted sequence were marked in grey highlighted. The mutations were detected by T7E1 assay together with PCR-sequencing or TA cloning-sequencing.