Cas9-Mediated Gene-Editing in the Malaria Mosquito Anopheles stephensi by ReMOT Control

Abstract

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for nonspecialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.

CRISPR/Cas9-based genome editing in the silverleaf whitefly (Bemisia tabaci)

“…In this report, we demonstrated heritable CRISPR/Cas9 gene editing in B. tabaci using ReMOT Control by changing the ovary targeting ligand from P2C to BtKV. P2C was derived from the yolk protein of Drosophila melanogaster and was used for gene editing in Aedes aegypti (10) and Anopheles stephensi (9). In our experiments, P2C did not generate knockouts in B.…”

“…When females <24 hpe were injected, editing rates in the offspring were >20% in some replicates, high enough for lineage screening by PCR to identify mutant individuals in the case of a gene without an obvious morphological phenotype. Across all replicates using 4 µg/mL saponin or less, injection of approximately 12 females was enough to obtain at least one edited offspring, an editing efficiency almost two-fold greater than observed in mosquitoes (9,10).…”

“…This resulted in high efficiency gene editing. In mosquitoes use of an endosomal escape reagent is critical to the success of ReMOT Control (9, 10), and saponin was shown to be highly effective (9). However, in B. tabaci we found a chemical endosomal escape reagent was unnecessary, and in fact saponin was detrimental to the process.…”

“…To generate mutants, the injection mixture consisted of the RNP complex of BtKV-Cas9 (3 mg/mL) with a mix of all sgRNAs (3 mg/mL) at a 1:3 molar ratio, and 1/3 rd the volume of saponin [4, 8, or 16 µg/mL] as an endosome escape reagent (or buffer as a control) (9,10). Five sgRNA multiplexed were used at a concentration of 250 ng/µL each and 1.25 µg/µL total.…”

“…A method has recently been developed called "ReMOT Control" (Receptor-Mediated Ovary Transduction of Cargo) which circumvents the need to inject embryos, but rather uses a small ovary-targeting peptide to directly transduce the Cas9 ribonucleoprotein complex (RNP; the Cas9 protein complexed with a guide RNA) into the developing ovaries upon in-jection into the hemolymph of adult female insects. ReMOT Control has been shown to efficiently edit the germline of the mosquitoes Aedes aegypti and Anopheles stephensi (9,10).…”