Abstract
CRISPR-based gene drives offer promising means to reduce the burden of pests and vector-borne diseases. These techniques consist of releasing genetically modified organisms carrying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modifications. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer fitness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes.
Highlights
CRISPR-based gene drives offer promising means to reduce the burden of pests and vectorborne diseases
Our experiments show that a single low frequency release of males carrying the anti-CRISPR construct is highly effective in preventing population suppression, despite a reduced fitness associated with the anti-drive
Gene drive inhibition is achieved via protein–protein interaction, rather than DNA cleavage of drive-specific sequences, bypassing potential unintended effects of genome editing alterations caused by alternative Cas9–gRNA or gRNA-only reversal strategies[15,17,18,19]
Summary
CRISPR-based gene drives offer promising means to reduce the burden of pests and vectorborne diseases. The management of vector and pest populations using nuclease-based gene drives is becoming a realistic possibility, after the recent proof-of-principle demonstrations of genetic control technologies based on the broadly applicable CRISPR–Cas nucleases[1] These technologies rely on the release of genetically engineered individuals that can rapidly propagate genetic constructs through wild populations together with the linked genetic modifications (e.g., knockout of sex determination[2] or fertility genes3) or the introduction of genetic cargos (e.g., pathogen-killing molecules designed to block the development of parasites within the vector[4]). Guide RNAonly systems developed in Drosophila showed capacity to inactivate or replace gene drives in caged populations[19] These strategies may offer the option to replace the drive with one or few “refractory alleles” or even restore the wild-type population, there are several complications attributable to the DNAcleaving nature of the reversal that remain to be addressed, such as the formation and selection of resistant alleles and/or genomic rearrangement at the drive locus targeted by the reversal nuclease. We generated an A. gambiae transgenic line expressing the AcrIIA4 protein from the Listeria monocytogenes prophage[20,21,22,23], and assessed its ability to both inhibit superMendelian inheritance of different driving constructs and prevent gene drive-mediated suppression of caged malaria mosquito populations
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