The increasing prevalence of insecticide resistance and the ongoing global burden of vector-borne diseases have encouraged new efforts in mosquito control. For Aedes aegypti, the most important arboviral vector, integration rates achieved in Cas9-based knock-ins so far have been rather low, highlighting the need to understand gene conversion patterns and other factors that influence homology-directed repair (HDR) events in this species. In this study, we report the effects of sequence mismatches or donor template forms on integration rates. We found that modest sequence differences between construct homology arms [DNA sequence in the donor template which resembles the region flanking the target cut] and genomic target comprising 1.2% nucleotide dissimilarity (heterology) significantly reduced integration rates. While most integrations (59–88%) from plasmid templates were the result of canonical [on target, perfect repair] HDR events, no canonical events were identified from other donor types (i.e. ssDNA, biotinylated ds/ssDNA). Sequencing of the transgene flanking region in 69 individuals with canonical integrations revealed 60% of conversion tracts to be unidirectional and extend up to 220 bp proximal to the break, though in three individuals bidirectional conversion of up to 725 bp was observed.

Aedes aegypti, the yellow fever mosquito, is the main vector of several major pathogens including yellow fever, dengue, Zika and chikungunya viruses. Classical mosquito control strategies, mainly utilizing insecticides, have had success in controlling other mosquito vectors in recent years, but are much less useful against Ae. aegypti, and even these methods are threatened by rising insecticide resistance This has stimulated interest in new mosquito control mechanisms, notably genetic systems such as gene drives. However, the development of CRISPR/Cas9 gene drive systems has faced challenges such as low inheritance biasing rate, the emergence of resistance alleles, and the possibility of spreading beyond the intended population. Here, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 in the germline to find an expression timing more conducive to homing. We also created a separate multiplexing (targeting multiple different sites within the target gene) sgRNA-expressing homing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene to limit the consequences of resistance alleles. This creates a split gene drive such that one part does not drive, allowing control over geographic spread and temporal persistence. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element was driven through a cage trial population such that carrier frequency of the element increased from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.

Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.

The Lepidoptera are an insect order of cultural, economic, and environmental importance, representing ∼10% of all described living species. Yet, for all but one of these species (silkmoth, Bombyx mori ), the molecular genetics of how sexual fate is determined remains unknown. We investigated this in the diamondback moth ( Plutella xylostella ), a globally important, highly invasive, and economically damaging pest of cruciferous crops. Our previous work uncovered a regulator of male sex determination in P. xylostella — PxyMasc , a homolog of B. mori Masculinizer —which, although initially expressed in embryos of both sexes, is then reduced in female embryos, leading to female-specific splicing of doublesex . Here, through sequencing small RNA libraries generated from early embryos and sexed larval pools, we identified a variety of small silencing RNAs (predominantly Piwi-interacting RNAs [piRNAs]) complementary to PxyMasc , whose temporal expression correlated with the reduction in PxyMasc transcript observed previously in females. Analysis of these small RNAs showed that they are expressed from tandemly arranged, multicopy arrays found exclusively on the W (female-specific) chromosome, which we term “ Pxyfem ”. Analysis of the Pxyfem sequences showed that they are partial complementary DNAs (cDNAs) of PxyMasc messenger RNA (mRNA) transcripts, likely integrated into transposable element graveyards by the noncanonical action of retrotransposons (retrocopies), and that their apparent similarity to B. mori feminizer more probably represents convergent evolution. Our study helps elucidate the sex determination cascade in this globally important pest and highlights the “shortcuts” that retrotransposition events can facilitate in the evolution of complex molecular cascades, including sex determination.