Aedes cadherin receptor that mediates Bacillus thuringiensis Cry11A toxicity is essential for mosquito development
Fig 3
CRISPR-Cas9 mediated EGFP tagging at the 3’ end of the last coding exon of AaeCad gene results in an in frame fusion.
A. sgRNA (in red) and PAM sequences (in blue) were designed before the stop codon (in purple) on the last exon of AaeCad gene. B. This donor plasmid for homologous recombination (HR) contains 1000bp of left arm, 720 bp of EGFP ORF and 1000bp of right arm. To prevent Cas9 cleavage on the left arm in the plasmid, a silent mutation (CGG → CTG) was introduced into the PAM sequence on the left arm. C. When the homologous recombination occurred, the EGFP was introduced immediately after the AaeCad gene with the aid of the left and right arms. D. The sequencing results of gDNA showed the AaeCad gene had been successfully tagged with EGFP and no mutation was introduced into the AaeCad gene except for the silent mutation that was intentionally introduced.
