Fig 1.
Schematic representation of the transgene in Ae. aegypti line PUbB2 P61 and the ECFP gene depicting sg35 and sg13 target sites.
A single copy of the transgene is shown. The protospacer adjacent motifs (PAM) are indicated in red. Abbreviations: pB left, pB right = piggyBac transposon left or right arm; svA = SV40 polyA signal; ECFP = cyan fluorescent protein gene; 3xP3 = eye-specific synthetic promoter; attR, attL = right or left PhiC31 attachment site; DsRed = red fluorescent protein gene; v5B2 = Flockhouse virus B2 gene; PUb = Ae. aegypti poly-ubiquitin promoter.
Fig 2.
Flowchart showing generation and identification of ECFP knockout mutants based on transgenic PUbB2 P61 x HWE hybrids.
Five groups of Cas9/sgRNA constructs were each micro-injected into ~500–700 double-hemizygous embryos (G0). All surviving G0 individuals were outcrossed to HWE and pooled. All G1 individuals of each pool were screened for the ECFP knockout phenotype. Pools containing individuals with ECFP knockout phenotype were again outcrossed to HWE to generate G2. DNA was extracted and sequenced from G0 adults and from G1 and G2 larvae showing ECFP knockout phenotype and also from groups of G1 larvae, which did not show a mutant phenotype.
Table 1.
Transformation data of PUbB2 P61 x HWE embryos injected with different CRISPR/Cas9 constructs
Fig 3.
Eye marker expression in PUbB2 P61 x HWE mosquitoes before and after CRISPR/Cas9 mediated ECFP knockout.
Eyes were viewed under a fluorescent stereo microscope (Leica M205) equipped with DsRed (A, C, E) or ECFP (B, D, F) specific filter sets. (A, B) Eyes of a PUbB2 P61 x HWE female. (C, D) Eyes of a (PUbB2 P61 x HWE) P41 female (G1) originating from an embryo which had been injected with in vitro transcribed RNAs encoding Cas9 and two ECFP targeting sgRNAs, sg13 and sg35. (E, F) Eyes of a HWE female. (G) Eyes of the HWE female under bright field.
Table 2.
Proportion of different eye marker phenotypes among G1 and G2 larvae of pools P41, P49, P55, and family F82.
Fig 4.
CRISPR/Cas9 mediated indels in the ECFP gene of PUBB2 P61 x HWE hybrids.
(A) Sanger-sequencing trace data showing the regions of the indels in mutants P41, P49, P55, and F82. Arrows indicate the cleavage site; the sequence of guide RNA sg35 is shadowed. (B) Nucleotide sequence alignments showing indels. The ECFP nucleotide sequence is shown at the top with the sg35 target site in bold-type and the PAM sequence in red. Deletions are shown as dashes. cDNA fragments spanning each target site were PCR-amplified using genomic DNA from pooled larvae as template and cloned into the pCR4-TOPO TA vector. At least 10 cDNA clones per PCR amplicon were sequenced.