Table 1.
CRISPR Cas9/sgRNA Act4 mutagenesis in Cx. quinquefasciatus and Ae. aegypti.
Fig 1.
AeAct4 gene disruption by homology-directed repair results in female-specific recessive lack of flight ability.
(A) Injected Cas9 protein and sgRNAs targeting AeAct4 induce a double stranded break (DSB) at the target site. A donor plasmid (AGG1070) having 2kb homology arms corresponding to the immediate upstream and downstream regions of the outermost cut sites was injected as a template for HDR. A 3xP3-mCherry-SV40 cassette serves as a marker for integration. Male-specific alternative splicing incorporates a number of early start and stop codons (marked by blue x) which ablate AeAct4 translation. Green arrows represent primer pair used for amplicons shown in (c). (B) 50 flightless females were identified out of a total of 156 marker-positive female progeny (32%). (C) Molecular confirmation of 6 flying and 4 flightless mCherry positive female adults, individually analyzed by PCR using the primers indicated in (A). AeAct4hdr1 and WT amplicons were generated. Based on this PCR assay, of the marker-positive females analyzed, all fliers were heterozygous for AeAct4hdr1 and all flightless females were homozygous.
Table 2.
HDR mediated CRISPR Cas9/sgRNA AeAct4 mutagenesis in Ae. aegypti.
Table 3.
156_161delTGCCTA ssODN HDR CRISPR/Cas9/sgRNA AeAct4 mutagenesis in Ae. aegypti.
Table 4.
Potential antimorphic mutations from ssODN HDR CRISPR Cas9/sgRNA AeAct4 mutagenesis in Ae. aegypti.
Fig 2.
Transmission electron microscope images of the indirect flight muscles of Ae. aegypti female adults arranged within the thorax.
Filaments in the myofibrils appear to be disrupted in AeAct4hdr1 homozygotes (A, arrows and inset) but not in AeAct4hdr1 heterozygotes (B, inset) or WT (C, inset). Individual myofibrils are indicated with double-headed arrows, and mitochondria (Mi) in the muscle fiber cells are clearly visible. Insert scale bars = 200 nm.