Table 3. 156_161delTGCCTA ssODN HDR CRISPR/Cas9/sgRNA AeAct4 mutagenesis in Ae. aegypti.
| 17 flightless and 17 flying female G1s were processed for AeAct4 sanger sequencing | ||||||||
|---|---|---|---|---|---|---|---|---|
| Injected components | No. Injected | G0 survivors (%) | G0 mosaic females (%) | G1 flightless female adults/total female G1s (%) | G0 founders (%) | 156_161delTGCCTA AeAct4 mutant flightless females/total G1 females (%) | AeAct4 mutant flightless females/total flightless female G1s sequenced (%) | AeAct4 mutant flying females/total flying female sequenced (%) |
| LA1422 ssODN AeAct4 donor oligo, AeAct4 sgRNA 2 | 580 | 187 (32.2) | 5 (5.9) | 27/1978 (1.4) | ≥2 (1) | 6/1978 (0.3) | 14/17 (82) | 0/17 (0) |
The 156_161delTGCCTA 6bp deletion found in Cx. quinquefasciatus was replicated in Ae. aegypti via ssODN HDR CRISPR/Cas9 mutagenesis of an exu-Cas9 line [33]. Potential flightless G0 mosaics and G1 mutants. 17 flightless and 17 flying G1s were selected for Sanger sequencing of AeAct4. All flying G1s had wild type sequence. 14 out of the 17 selected flightless G1s had in-frame indels, 6 of which showed the intended 156_161delTGCCTA deletion. These 6 individuals originated from 2 different G0 pools. G0s were crossed in pools; hence, the minimum value for founders. To assess the rate of targeted mutation, rather than all mutation, G0s were only considered founders if they resulted in G1s with successful HDR events reproducing the donor template 6bp deletion; founders of other mutagenesis events are not included. sgRNA concentration was 40 ng/μl and donor oligo 125 ng/μl.