Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti

Abstract

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.

Keywords: Aedes aegypti; CRISPR; cas9; germline; mutagenesis.

Fig. 1.

Rationally chosen, native promoters drive strong expression of Cas9. Log₂ RPKM expression values for AAEL010097, AAEL007097, AAEL007584, AAEL005635, AAEL003877, and AAEL006511 were plotted across development. Samples include, from left to right: testis; male carcasses (lacking testes); carcasses of females before blood feeding (NBF), and at 12-, 24-, 36-, 48-, and 72-h postblood meal (PBM); ovaries from NBF females and at 12-, 24-, 36-, 48-, and 72-h PBM; embryos from 0–2 h through 72–76 h; whole larvae from first, second, third, and fourth instar; male pupae; female pupae (A). Genome browser snapshots of AAEL010097, AAEL007097, AAEL007584, AAEL005635, AAEL003877, and AAEL006511, including expression tracks for both 72-h PBM ovaries and testes. The light gray box indicates coding sequence; blue box represents promoter element with length indicated in bp; y axis shows the expression level based on raw read counts (B). Schematic representation of the piggybac-mediated Cas9 construct including the S. pyogenes Cas9-T2A-eGFP gene driven by selected promoters (blue), dsRed expressed under the control of the Opie2 promoter, which serves as a transgenesis marker. NLS represent nuclear localization signal (C). Confocal images using white light or GFP-filtering of transgenic Cas9 line ovaries (D). (Magnification, 200×.)

Fig. 2.

Severe mutant phenotypes caused by CRISPR/Cas9 mediated disruption. Larva, pupae, and adult phenotypes of wild-type, kh, white, yellow, and ebony mutant G₁s, respectively, with clearly distinguishable eye (kh and white) and cuticle pigment (yellow and ebony) defects (A). Embryo phenotypes of wild-type (Left) and yellow mutants (Right) (B). Pupae and adult scanning electron microscopy images of the head of the deformed G₀ mutants with three compound eyes (Ce), three maxillary palps (Mp), furrowed eyes, and deformed mouthparts (arrows) (C). Pupae and adult scanning electron microscopy images of the head of the sine oculis G₀ mutants. Arrows point to the ectopic eyes (D). Images of pupae and adult wings of vestigial G₀ mutants. Arrows point to pronounced wing, halteres, and forked wing defects (E). (Magnifications: whole-body images, 20×; Insets, 100×; SEM images, 150×.)

Fig. 3.

Single injections of multiplexed sgRNAa robustly generate double- and triple-mutant mosquitoes. Larva, pupae, and adult G₁ phenotypes for double-mutants, including: yellow body and white eyes (yellow/white), a mixture of yellow and dark body (yellow/ebony), dark body and white eyes (ebony/white), and one triple-mutant, which is a phenotypic mixture of yellow and dark body and white eyes (yellow/ebony/white). The striking differences between wild-type and mutant larva, pupae and adult are highlighted. (Magnifications: whole-body images, 20×; Insets, 100×.)

Fig. 4.

Highly efficient site-specific integration via CRISPR-mediated HDR. Schematic representations of the white locus and white-donor construct (A), and the kh locus and kh-donor construct (B). Exons are shown as boxes, coding regions are depicted in black and the 5′ and 3′ UTRs in gray. Locations and sequences of the sgRNA targets are indicated with the PAM shown in yellow. Black arrows indicate approximate positions and directions of the oligonucleotide primers used in the study. The donor plasmids (blue) express fluorescent eye marker (3xp3-DsRed) inserted between regions of homology from the white and kh locus, respectively (A and B). Gene amplification analysis confirms site-specific integration of the white-donor construct into the white locus using combinations of genomic and plasmid donor-specific primers (933Cms3/933Cms4 expected 349 bp, and 933Cms5/933Cms6 expected 533 bp) (C), and also confirms the integration of the kh-donor construct into the kh locus using combinations of genomic and plasmid donor specific primers (924ms3/924ms4 expected 525 bp, and 924ms5/924ms6 expected 745 bp) with no amplification in wild-type (D). WT represents wild-type, WD represents knockin with white-donor, KHD represents knockin with kh-donor. (Magnification: 20×.)