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. 2016 May 3;6:25199. doi: 10.1038/srep25199

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.