Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.

CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.

Wolbachia pipientis is an insect endosymbiont known to limit the replication of viruses including dengue and Zika in their primary mosquito vector, Aedes aegypti. Wolbachia is being released into mosquito populations globally in a bid to control the diseases caused by these viruses. It is theorized that Wolbachia’s priming of the insect immune system may confer protection against subsequent viral infection. Other hypotheses posit a role for competition between Wolbachia and viruses for host cellular resources. Using an A. aegypti cell line infected with Wolbachia, we tested the effects of targeting siRNAs against the major innate immune pathways on dengue virus loads. We show that while Wolbachia infection induces genes in the Toll, JAK/STAT and RNAi pathways, only reduced expression of RNAi leads to a rebound of dengue virus loads in Wolbachia-infected cells. The magnitude of the effect explained less than 10% of the total DENV load, demonstrating that blocking must be dependent on other factors in addition to the expression of RNAi. The findings bode well for the long-term stability of blocking given that immunity gene expression would likely be highly plastic and susceptible to rapid evolution.

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bSterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase recently recognized as an antiviral factor that acts by depleting dNTP availability for viral reverse transcriptase (RT). SAMHD1 restriction is counteracted by the human immunodeficiency virus type 2 (HIV-2) accessory protein Vpx, which targets SAMHD1 for proteosomal degradation, resulting in an increased availability of dNTPs and consequently enhanced viral replication. Nucleoside reverse transcriptase inhibitors (NRTI), one of the most common agents used in antiretroviral therapy, compete with intracellular dNTPs as the substrate for viral RT. Consequently, SAMHD1 activity may be influencing NRTI efficacy in inhibiting viral replication. Here, a panel of different RT inhibitors was analyzed for their different antiviral efficacy depending on SAMHD1. Antiviral potency was measured for all the inhibitors in transformed cell lines and primary monocytederived macrophages and CD4؉ T cells infected with HIV-1 with or without Vpx. No changes in sensitivity to non-NRTI or the integrase inhibitor raltegravir were observed, but for NRTI, sensitivity significantly changed only in the case of the thymidine analogs (AZT and d4T). The addition of exogenous thymidine mimicked the change in viral sensitivity observed after Vpx-mediated SAMHD1 degradation, pointing toward a differential effect of SAMHD1 activity on thymidine. Accordingly, sensitivity to AZT was also reduced in CD4؉ T cells infected with HIV-2 compared to infection with the HIV-2⌬Vpx strain. In conclusion, reduction of SAMHD1 levels significantly decreases HIV sensitivity to thymidine but not other nucleotide RT analog inhibitors in both macrophages and lymphocytes. Sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) is a recently identified human immunodeficiency virus type 1 (HIV-1) host restriction factor that limits retroviral replication at the reverse transcription stage of the viral life cycle (1-5). SAMHD1 functions as a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that regulates the intracellular pool of dNTPs (6). It restricts HIV-1 infection in immune cells of myeloid lineage and in quiescent CD4-positive T lymphocytes (1, 2, 4). SAMHD1 reduces cellular dNTP levels to concentrations below the threshold required for reverse transcription of the viral RNA genome into DNA (4, 5). SAMHD1 is counteracted by the retroviral Vpx protein that is encoded by simian immunodeficiency virus (SIV) and HIV-2, but this gene is lacking from the HIV-1 and feline immunodeficiency virus (FIV) genomes (7). However, and despite the lack of Vpx function, HIV-1 is still able to replicate in noncycling myeloid cells, albeit at low levels (7).Most current standard three-drug antiretroviral regimens involve RT inhibitors combined with a protease inhibitor. [EFV], and etravirine). NRTI are phosphorylated to their triphosphate form to act as competitive inhibitors of HIV RT. In contrast, NNRTI bind at...