Concerning RNA-guided gene drives for the alteration of wild populations

Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology. DOI: http://dx.doi.org/10.7554/eLife.03401.00

Harnessing gene drive

© 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. When scientists alter the genome of an organism, we typically reduce its ability to reproduce in the wild. This limitation has prevented researchers from rendering wild insects unable to spread disease, programing pests to ignore our crops, using genetics to precisely remove environmentally damaging invasive species, and much more. Gene drive occurs when a vertically transmitted genetic element reliably spreads through a population over generations despite providing no reproductive advantage to each host organism. Until recently, scientific efforts to take advantage of this natural phenomenon achieved only limited success. The advent of CRISPR genome editing has dramatically accelerated efforts to harness gene drive. Small groups of scientists may now be capable of unilaterally altering entire wild populations, and through them, the shared environment. Determining whether, when, and how to develop gene drive interventions responsibly will be a defining challenge of our time. Here we describe capabilities, safeguards, applications, and opportunities relevant to gene drive technologies

A transgenic tool to assess Anopheles mating competitiveness in the field

Abstract Background Malaria parasites, transmitted by the bite of an anopheline mosquito, pose an immense public health burden on many tropical and subtropical regions. The most important malaria vectors in sub-Saharan Africa are mosquitoes of the Anopheles gambiae complex including An. gambiae (sensu stricto). Given the increasing rates of insecticide resistance in these mosquitoes, alternative control strategies based on the release of genetically modified males are being evaluated to stop transmission by these disease vectors. These strategies rely on the mating competitiveness of release males, however currently there is no method to determine male mating success without sacrificing the female. Interestingly, unlike other insects, during mating An. gambiae males transfer their male accessory glands (MAGs) seminal secretions as a coagulated mating plug which is deposited in the female atrium. Results Here we exploit this male reproductive feature and validate the use of a MAG-specific promoter to fluorescently label the mating plug and visualize the occurrence of insemination in vivo. We used the promoter region of the major mating plug protein, Plugin, to control the expression of a Plugin-tdTomato (PluTo) fusion protein, hypothesizing that this fusion protein could be incorporated into the plug for sexual transfer to the female. Anopheles gambiae PluTo transgenic males showed strong red fluorescence specifically in the MAGs and with a pattern closely matching endogenous Plugin expression. Moreover, the fusion protein was integrated into the mating plug and transferred to the female atrium during mating where it could be visualized microscopically in vivo without sacrificing the female. PluTo males were equally as competitive at mating as wild type males, and females mated to these males did not show any reduction in reproductive fitness. Conclusion The validation of the first MAG-specific promoter in transgenic An. gambiae facilitates the live detection of successful insemination hours after copulation has occurred. This provides a valuable tool for the assessment of male mating competitiveness not only in laboratory experiments but also in semi-field and field studies aimed at testing the feasibility of releasing genetically modified mosquitoes for disease control

Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases

<div><p><i>Anopheles gambiae</i>, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene <i>TEP1</i> using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several <i>TEP1</i> mutant <i>A. gambiae</i> lines. These mutations inhibited protein production and rendered <i>TEP1</i> mutants hypersusceptible to <i>Plasmodium berghei</i>. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control.</p> </div

TALEN mutagenesis of the <i>TEP1</i> gene.

<p>A: Fragment from the <i>TEP1</i> gene showing the target site of the TALEN pair. Nucleotides bound by each TALEN are underlined, TALEN repeats are color-coded to show repeat/nucleotide specificity. The <i>Nco</i>I restriction site centrally located at the TALEN cleavage site is highlighted. Inset: scheme of the entire TEP1 protein showing the location of TALEN-induced mutations (SP: signal peptide; CUB: CUB domain interrupted by the TED: thioester domain; the star indicates the position of the thioester site). B: PCR assay to identify <i>TEP1</i> mutant mosquitoes. A PCR product spanning the TALEN target site is generated from individual mosquitoes (small larva or a leg from a living adult) and incubated with <i>Nco</i>I. Full cleavage of the PCR product (w) denotes a wild-type individual. Partial cleavage (h) denotes a heterozygous <i>TEP1</i> mutant. Absence of cleavage (H) corresponds to a homozygous <i>TEP1</i> mutant. C: TALEN-induced mutations in the TEP1 gene. Left and right TALEN target nucleotide sequences are shown in green and blue respectively, with the 15bp spacer sequence between the TALENs in black. The <i>Nco</i>I restriction site is highlighted in orange. Deletions are designated by a red dash or by Δ+number of missing bases. Insertions are shown in lowercase red letters. Uppercase red letters correspond to natural polymorphisms between multiple <i>TEP1</i> alleles.</p

<i>TEP1</i> mutant mosquitoes are hypersusceptible to <i>P. berghei</i>.

<p>Mosquito females from five different homozygous mutant mosquito lines and from control parental lines were offered a blood meal on a <i>P. berghei</i>-infected mouse. Seven days after infection, the midgut was dissected and the number of oocysts developing in each midgut was evaluated. The statistical significance of differences in mean parasite numbers was measured with a Mann-Whitney test (mutant versus control) and with a Kruskall-Wallis test followed by Dunn’s post-test (to compare all groups in [d]). (a) M^∆T mosquitoes are compared to the two parental, non-mutant TALEN lines. (b) 4 different mutant lines are compared to the parental mosquito line that initially served to produce TALEN transgenic lines. This control line was verified to show the same level of susceptibility to <i>P. berghei</i> as the two TALEN daughter lines (not shown). In this experiment, three mutant lines showed significantly elevated parasite numbers compared to the control while the M^∆ct2 line did not, presumably due to a different physiological condition of this mosquito culture. In two independent experiments (c, d), we used mosquitoes of different genotypes marked with distinct fluorescence markers and cultured the larvae together in the same water to eliminate potential confounding factors due to rearing conditions. On the day of dissection, genotypes were separated on the basis of fluorescence. The same M^∆ct2 line shows significantly elevated parasite numbers. (d) Heterozygous M^∆ct2 mosquitoes are compared to control and homozygous M^∆ct2 mosquitoes: the susceptibility phenotype of the heterozygote is intermediate. (e) Control mosquitoes of the parental line, mosquitoes of the parental line injected with <i>TEP1</i> double-stranded RNA, and homozygous <i>TEP1</i> mutant mosquitoes of the M^∆T line are compared. TEP1 mutant and dsRNA-injected mosquitoes show comparable susceptibility to <i>P. berghei</i>.</p

TEP1 mutant proteins.

<p>A: Fragment of the TEP1 protein encompassing the TALEN-induced mutations is shown for the wild-type (WT) and for those mutants that we maintain as homozygous mosquito lines (M^∆T, M^∆MV, M^∆3+6, M^∆ct1 and M^∆ct2). Gaps in the protein sequence denote amino acid deletions. Inserted exogenous amino acids are shown in red. Nonsense amino acids followed by a stop codon (*), resulting from frame-shift mutations, are shown in blue. B: Immunoblots to evaluate the presence of mutant TEP1 protein in whole mosquito extract (top panels) or hemolymph. Hemolymph prophenoloxidase (PPO) serves as a loading control. In the control samples, both TEP1 full-length (full) and C-terminal fragment (cleaved) are visible. Cross-reacting background bands, some running in close proximity to TEP1 fragments, are marked on the left with ‘>’ signs.</p

CRISPR-mediated germline mutagenesis for genetic sterilization of Anopheles gambiae males

Abstract Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile insect technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth (zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors