2.3 Tissue specific mutagenesis using tsCRSPR/Cas9

tsCRISPR based mutagenesis was performed as described, by crossing driver lines containing UAS-Cas9 and Gal4 under control of different tissue specific promoters with flies expressing sgRNAs (Port et al., 2020). For establishing eye-specific tsCRISPR sgRNA directed against norpA (VDRC ID: 341777) was used and mutagenesis efficiency was tested by immunoblot analysis and ERG measurements (see results). In our study we focused further on Rab3 and RabX2. Efficient gene disruption by the sgRNA line directed against Rab3 was shown previously (Allen et al., 2021; Sachidanandan et al., 2023). To verify efficient gene disruption for RabX2, we employed T7 endonuclease (NEB, # M0689L) digestion of hybridized PCR amplified genomic regions covering the sgRNA target sites of the RabX2 gene (ALLin™ Mega HiFi Mastermix (highQu, Cat#HLM0201), forward primer: CGA​CTT​GAC​GAT​GAG​CCA​CTT; reverse primer: CAC​ATG​GCG​CCG​TAT​CTC​CTT). The method relies on the characteristic of T7 endonuclease to digest DNA at base pair mismatches. Mutations induced by tsCRISPR are indicated by appearance of DNA bands smaller than the PCR amplificat. We amplified DNA obtained from single eyes of RabX2 gene disruption flies and found that 10 out of 10 eyes contained mutations in the amplified DNA.