Expanding the flexibility of genome editing approaches for population control of the malaria mosquito

Discovery and adaptation of CRISPR-Cas systems for genome editing have allowed us to gain an efficient and yet simple tool for genetic manipulation in various fields of molecular biology and biotechnology. One of the most promising applications is the use of CRISPR-Cas9 endonuclease for gene drive systems as a population control strategy for various insect pests of medical and agricultural importance. Use of CRISPR-Cas9 endonuclease in gene drive applications has shown great promise in the laboratory, particularly for the control of Anopheles gambiae, the major vector of malaria. However, the performance of such gene drives can be limited by the range of available target sequences and by a propensity of existing endonuclease formulations to generate resistant mutations that hinder the gene drive’s efficiency. To expand the flexibility of gene drive systems, computational analysis was performed to identify additional Cas9 orthologs and their specificities that could usefully augment the targeting range of endonuclease-based gene drives. Two alternative variants of CRISPR-Cas endonucleases found in the bacterial species Lactobacillus rhamnosus and Bacteroides fragilis were assessed for their potential to expand the targeting space in the genome Anopheles gambiae. In addition, a computational tool was developed that evaluates neighbouring sequences to the target site to measure both its likely functional constraint and its likely propensity for DNA repair that could generate in-frame alleles. Using this approach we were able to generate a prioritized list of Anopheles gambiae target sites for gene drive applications that are less likely to be compromised by resistant alleles.Open Acces

Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance

Homing-based gene drives use a germline source of nuclease to copy themselves at specific target sites in a genome and bias their inheritance. Such gene drives can be designed to spread and deliberately suppress populations of malaria mosquitoes by impairing female fertility. However, strong unintended fitness costs of the drive and a propensity to generate resistant mutations can limit a gene drive’s potential to spread. Alternative germline regulatory sequences in the drive element confer improved fecundity of carrier individuals and reduced propensity for target site resistance. This is explained by reduced rates of end-joining repair of DNA breaks from parentally deposited nuclease in the embryo, which can produce heritable mutations that reduce gene drive penetrance. We tracked the generation and selection of resistant mutations over the course of a gene drive invasion of a population. Improved gene drives show faster invasion dynamics, increased suppressive effect and later onset of target site resistance. Our results show that regulation of nuclease expression is as important as the choice of target site when developing a robust homing-based gene drive for population suppression

nkran/guido: v0.1.4

CRISPR sgRNA search and evaluation Python package

Analysis of the Genetic Variation of the Fruitless Gene within the Anopheles gambiae (Diptera: Culicidae) Complex Populations in Africa

Targeting genes involved in sexual determinism, for vector or pest control purposes, requires a better understanding of their polymorphism in natural populations in order to ensure a rapid spread of the construct. By using genomic data from An. gambiae s.l., we analyzed the genetic variation and the conservation score of the fru gene in 18 natural populations across Africa. A total of 34,339 SNPs were identified, including 3.11% non-synonymous segregating sites. Overall, the nucleotide diversity was low, and the Tajima’s D neutrality test was negative, indicating an excess of low frequency SNPs in the fru gene. The allelic frequencies of the non-synonymous SNPs were low (freq < 0.26), except for two SNPs identified at high frequencies (freq > 0.8) in the zinc-finger A and B protein domains. The conservation score was variable throughout the fru gene, with maximum values in the exonic regions compared to the intronic regions. These results showed a low genetic variation overall in the exonic regions, especially the male sex-specific exon and the BTB-exon 1 of the fru gene. These findings will facilitate the development of an effective gene drive construct targeting the fru gene that can rapidly spread without encountering resistance in wild populations

The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito

Gene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance, gene drives can rapidly introduce genetic traits even if these confer a negative fitness effect on the population. We have recently developed gene drives based on CRISPR nuclease constructs that are designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression, though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we observed a gradual decrease in its frequency that was accompanied by the spread of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations showed rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance in future gene drive applications

Dynamics of a population suppression gene drive construct over 25 generations.

<p><b>(A)</b> Design of the CRISPR-based gene drive construct and the relevant position of its target site within AGAP007280; <b>(B)</b> The proportion of individuals containing at least one copy of the gene drive in two replicate cages, monitored each generation for 25 generations. Black lines represent the observed frequencies in each of the two cage trials (CT1 and CT2), red line represents the predicted frequency according to the previous deterministic model that did not take into account target site resistance [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007039#pgen.1007039.ref011" target="_blank">11</a>]. Samples were taken for pooled PCR and sequencing analysis of the gene drive target site at G₂ and at G₁₂, whereby frequency of target site indels (shown figuratively as red bars) were revealed by their relative representation among the sequenced reads.</p

Comparison of observed data with model predicting frequencies of gene drive and resistance alleles.

<p><b>(A)</b> Expected genotype frequencies according to the model described in the text and considering the four following target site alleles: wild type (w), <i>CRISPR^h</i> gene drive (h), resistant and in-frame (r1), resistant and out of frame (r2). We used our best experimental estimates of the considered parameters: homing rate (<i>e</i>) as 0.984, the dominance of the fertility effect due to leaky somatic expression in females heterozygous (<i>w/h</i>) for the gene drive as 0.907, meiotic end-joining rate (γ_m) as 0.01, embryonic end-joining rate (γ_e) as 0.796. <b>(B)</b> Our observed gene drive frequencies were compared against model predictions using our best experimental estimates (solid black line) and using the best-fit value (0.70 cf 0.907) for dominance of the heterozygous fertility effect in females (dashed black line).</p

Target site mutations under positive selection are resistant to gene drive activity and restore function to the target female fertility gene.

<p><b>(A)</b> Individual females containing at least one copy of the gene drive (RFP+) were selected from the G₂₀ generation and the nature of the target allele was determined by PCR and sequencing. Each class of allele is shown with gene drive target sequence highlighted in red and PAM sequence underlined. <b>(B)</b> The fecundity of these females and transmission rates of the gene drive were measured and grouped according to allele class at the target site. <b>(C)</b> Each <i>CRISPR</i>^<i>h</i><i>/r</i> female was used to form a separate lineage and transmission of the gene drive was assessed in sons receiving a maternal copy of the gene drive. A smaller fraction of grandsons receiving a paternal copy of the gene drive were similarly assessed for gene drive transmission. Individual lineages assessed in all three generations are marked in red. † Of 58 mated females one (with deletion 207+AAAGTC) failed to produce eggs while another (202–TGAGGA) produced eggs that failed to hatch.</p

A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive