Table 2.
Efficiencies of correct replacements of target genes with the UDH expression cassette in multiplexed CRISPR/Cas9 using RNP complexes
| Target genes | gRNA | Colonies screened | Correct ΔgaaA | Correct Δ39114 | Correct ΔgaaX | All correct | Targeting efficiency (%) |
|---|---|---|---|---|---|---|---|
| gaaA | gERA-009 | 2 | 2 | – | – | 2 | 100 |
| gERA-010 | 5 | 5 | – | – | 5 | 100 | |
| gERA-009 + gERA-010 | 5 | 5 | – | – | 5 | 100 | |
| gaaA + 39114 | gERA-009 + gERA-015 | 4 | 3 | 1 | – | 1 | 25 |
| gERA-010 + gERA-016 | 7 | 1 | 2 | – | 0 | 0 | |
| gERA-009 + gERA-015+ | 8 | 1 | 3 | – | 1 | 13 | |
| gERA-010 + gERA-016 | |||||||
| gaaA + 39114 + gaaX | gERA-001 + gERA-009 + gERA-015 | 6 | 3 | 1 | 2 | 0 | 0 |
| gERA-002 + gERA-010 + gERA-016 | 6 | 1 | 1 | 4 | 1 | 17 | |
| gERA-001 + gERA-009 + gERA-015 + gERA-002 + gERA-010 + gERA-016 | 5 | 2 | 2 | 2 | 0 | 0 |
Two different crRNAs for each of the target genes were designed and tested in different combinations