Figure 2.

“unc-22” Co-CRISPR as a marker to indicate actively expressed Cas9. (A) Schematic of Co-CRISPR strategy to identify functional sgRNAs targeting avr genes. sgRNAs targeting avr-14 and avr-15 were co-injected with a functional unc-22 sgRNA, the Cas9 expression vector, and the rol-6 transformation marker. F1 rollers or twitchers were transferred to individual plates. The plates were allowed to starve, and then they were copied to plates containing 2 ng/ml ivermectin to identify CRISPR-Cas9-induced avr-14; avr-15 double mutants. (B) Indel sequences in avr-14; unc-22; avr-15 triple mutants. avr-15 isolate 15 carried different indels on each allele. Sequences labeled with a question mark could not be precisely determined. (C) Comparison of twitcher-based indel frequency and roller-based indel frequency.