Fig. 1.

D. melanogaster and B. mori U6 promoters do not support CRISPR-Cas9 editing in Sf9 cells. (A) Diagram showing generic CRISPR-Cas9 vectors encoding, (Left to Right) SpCas9 under the control of a baculovirus ie1 promoter, an sgRNA expression cassette that includes an insect species-specific U6 promoter and a targeting sequence cloning site consisting of two SapI recognition sites, and a puromycin-resistance marker under the control of baculovirus hr5 enhancer and ie1 promoter elements. (B) Diagram showing Sf-fdl gene structure and highlighting specific Cas9 targeting sequences (Table S1) and PCR primer sites. (C) CEL-I nuclease assay results obtained using genomic DNA from Sf9 cells edited with CRISPR-Cas9 vectors encoding various Sf-fdl targeting sequences (SfFDLt1, SfFDLt2, and SfFDLt3) (Table S1) under the control of either the DmU6:96Ab or the BmU6-2 promoter.