TABLE 2.
Comparison of efficiency of generating frameshift mutations from each category of gene disruption
| Category | Strain + sgRNA | Bacterial survival (%)a | Clones analyzed | Frameshift frequency (%) | Overall frequency of desired outcomes (%)b |
|---|---|---|---|---|---|
| Single gene disruption | WT + bla sgRNA | 10.6 | 8 | 87.5 | 9.3 |
| WT + arr sgRNA | 9.1 | 6 | 83.3 | 7.6 | |
| WT + eis2 sgRNA | 1.6 | 6 | 66.7 | 1.1 | |
| Simultaneous gene disruption | WT + (bla + arr) double sgRNA | 0.19 | 12 | 58.3c | 0.11 |
| Sequential gene disruption | Δbla-1 + arr sgRNA | 5.7 | 6 | 33.3 | 1.9 |
| Δbla-2 + arr sgRNA | 4.8 | 6 | 66.7 | 3.2 | |
| Δbla Δarr + eis2 sgRNA | 0.56 | 6 | 66.7 | 0.32 |
a
Bacterial survival (%) calculated from CFUs from Cas9-induced bacteria relative to CFUs in uninduced bacteria. Mean of three technical replicates indicated.
b
Overall frequency of desired frameshift outcomes calculated as (bacterial survival × frameshift frequency).
c
Frameshift frequency of both genes.