Table 1.
Mutation rates in pZH_p-gRNA_MMCas9-Nuclease- or pZH_p-gRNA_MMCas9-Nickase-transformed calli
| Paired gRNAs(MF ranka) | Target gene | Overhang | Distance of paired gRNAs (bp) | Cas9 paired nucleases |
Cas9 paired nickases |
|||
|---|---|---|---|---|---|---|---|---|
| RCb | AMF (%)c | RCb | AMF (%)c | |||||
| A1t | AB3b | DMC1A | 5′ | 99 | 24/24 | 54.1 | 1/24 | ND |
| (Low) | (High) | DMC1B | – | – | 23/24 | 70.4 | 0/24 | – |
| A2t | AB3b | DMC1A | 5′ | 71 | 24/24 | 85.4 | 23/24 | 56.2 |
| (High) | (High) | DMC1B | – | – | 24/24 | 75 | 0/24 | – |
| A2t-1 | AB3b-1 | DMC1A | 5′ | 71 | 24/24 | ND | ||
| A2t-2 | AB3b-2 | DMC1A | 5′ | 71 | 0/24 | – | ||
| AB4t | AB3b | DMC1A | 3′ | 37 | 24/24d | 84 | 2/24d | ND |
| (High) | (High) | DMC1B | 3′ | 37 | 24/24d | 79.4 | 1/24d | ND |
| AB4t | AB5b | DMC1A | 5′ | 11 | 23/24 | 47.9 | 14/24 | 18.7 |
| (High) | (Medium) | DMC1B | 5′ | 11 | 22/24 | 25.0 | 13/24 | 10.4 |
| AB4t | A6b | DMC1A | 5′ | 66 | 23/24 | 64.5 | 20/24 | 40.4 |
| (High) | (Medium) | DMC1B | – | – | 23/24 | 43.3 | 0/24 | – |
| AB4t | AB7b | DMC1A | 5′ | 167 | 23/24 | 43.1 | 1/24 | ND |
| (High) | (Low) | DMC1B | – | – | 23/24 | 37.6 | 0/24 | – |
| B1t | BA1b | CDKB2 | 5′ | 30 | 20/24 | 41.6 | 24/24 | 22.7 |
| CDKA2 | – | – | 20/24 | 33.3 | 0/24 | – | ||
| (Medium) | (Medium) | CDKB1 | – | – | 13/24 | 8.3 | 0/24 | – |
| CDKA1 | – | – | 5/24 | 2.0 | 0/24 | – | ||
| B2t | BA1b | CDKB2 | 5′ | 127 | 18/24 | 40.9 | 20/24 | 14.5 |
| CDKA2 | – | – | 18/24 | 29.1 | 0/24 | – | ||
| (Medium) | (Medium) | CDKB1 | – | – | 12/24 | 4.5 | 0/24 | – |
| CDKA1 | – | – | 2/24 | 2.0 | 0/24 | – | ||
The total number of sequenced clones is approximately 48 (16 clones × 3 lines).
a The rank of mutation frequency (MF) using single gRNA and Cas9 nuclease (Supplementary Table S1).
b Ratio of calli with mutations (RC) by CAPS analysis of gRNA which recognizes on- and off-target sites (AB3b, AB4t and BA1b).
c Average mutation frequency (AMF) of three lines (Supplementary Fig. S1).
d CAPS analysis of AB3b site.