Table 3 Comparison of off target mutation in both mouse cell lines and mice induced by tru-RGNs and std-RGNs.

From: Efficient generation of FVII gene knockout mice using CRISPR/Cas9 nuclease and truncated guided RNAs

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 ± 0.8 a

13.1 ± 0.3 a

0 a

0 a

OT1–2

AGGATGGGCCAACTCACTCC TGG

GATGGGCCAACTCACTCC TGG

6.6 ± 0.2 a

6.2 ± 0.3 a

0 a

0 a

OT1–3

AAGCCCTCCTAACTCACTCC CAG

GCCCTCCTAACTCACTCC CAG

0 a

0 a

0 a

0 a

OT1–4

AAGCCATCCAAACTCACTCC TGG

GCCATCCAAACTCACTCC TGG

21.4 ± 0.6 a

20.3 ± 1.0 a

0 a

0 a

OT1–5

ACTGTGTGCCACCTCACTCC TGG

TGTGTGCCACCTCACTCC TGG

2.1 ± 0.4 a

0 b

0 a

0 a

FVII site 2 (on-target)

GCGTGCCAACTCACTCCTGG AGG

GTGCCAACTCACTCCTGG AGG

    

OT2–1

GCTGGCAAACTCACTCCTGG AAG

TGGCAAACTCACTCCTGG AAG

4.4 ± 0.4 a

3.0 ± 0.1 b

0 a

0 a

OT2–2

GGCTGCCACCTCACTCCTGG AGG

CTGCCACCTCACTCCTGG AGG

0 a

0 a

0 a

0 a

OT2–3

GTGTGTCTACTCACTCCTGG GGG

GTGTCTACTCACTCCTGG GGG

0 a

0 a

0 a

0 a

OT2–4

CTGTCCCAATTCACTCCTGG TAG

GTCCCAATTCACTCCTGG TAG

0 a

0 a

0 a

0 a

OT2–5

AGGTACCAGCTCACTCCTGG GGG

GTACCAGCTCACTCCTGG GGG

8.5 ± 0.5 a

6.0 ± 0.3 b

0 a

0 a

FVII site 3 (on-target)

GGAGCTTTGGCCCGGCTCTC TGG

GCTTTGGCCCGGCTCTC TGG

    

OT3–1

GGGGCTTTGGCCAGGCTCTC AGG

GCTTTGGCCAGGCTCTC AGG

0 a

0 a

0 a

0 a

OT3–2

AGGGCCTTGGCCCGGCTCTC GGG

GCCTTGGCCCGGCTCTC GGG

3.7 ± 0.5 a

22.0 ± 0.6 b

0 a

29.3 ± 7.1 b

OT3–3

TAAACTTTGACCCGGCTCTC TAG

ACTTTGACCCGGCTCTC TAG

5.7 ± 0.4 a

5.2 ± 0.4 a

0 a

0 a

OT3–4

ACAGCTTTGGCCAGGCTCTC GAG

GCTTTGGCCAGGCTCTC GAG

6.2 ± 0.3 a

3.2 ± 0.2 b

0 a

0 a

OT3–5

ACAGCTTTGGCCAGGCTCTC GAG

GCTTTGGCCAGGCTCTC GAG

7.2 ± 0.1 a

5.6 ± 0.2

0 a

0 a

  1. 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.
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