Abundance of conserved CRISPR-Cas9 target sites within the highly polymorphic genomes of Anopheles and Aedes mosquitoes

Abstract

A number of recent papers report that standing genetic variation in natural populations includes ubiquitous polymorphisms within target sites for Cas9-based gene drive (CGD) and that these "drive resistant alleles" (DRA) preclude the successful application of CGD for managing these populations. Here we report the results of a survey of 1280 genomes of the mosquitoes Anopheles gambiae, An. coluzzii, and Aedes aegypti in which we determine that ~90% of all protein-encoding CGD target genes in natural populations include at least one target site with no DRAs at a frequency of ≥1.0%. We conclude that the abundance of conserved target sites in mosquito genomes and the inherent flexibility in CGD design obviates the concern that DRAs present in the standing genetic variation of mosquito populations will be detrimental to the deployment of this technology for population modification strategies.

Fig. 1. Sketch of the effect of quality filtering on the number of “good” targets/genes.

Genome-wide count of CRISPR-Cas9 targets (orange) and protein-coding genes (blue) is set to 100% each. During specificity filtering (GC content between 30 and 70% and no off-targets) and DRA filtering (DRA frequency <1%), the number of available targets drops well below 10% (Table 2). Nevertheless, ~90% of all protein-coding genes still contain at least one good target. Colored areas correspond to the values for the combined data of An. gambiae and An. coluzzii. The percentages are similar for Ae. aegypti (not shown). Source data are provided as a Source Data file.

Fig. 2. Frequency of genes with good targets.

The fraction of genes with good targets is dependent on the presence of low-frequency DRAs. When dismissing all targets with DRA frequencies > 0.0, the fraction of genes with good targets decreases with increasing sample size. Ignoring DRAs with frequencies below 1% in the dataset results in ~90% of genes having at least one good target in all datasets examined. VGL: Vector Genetics Laboratory, Ag1000G: The Anopheles gambiae 1000 Genomes Consortium. Source data are provided as a Source Data file.

Fig. 3. Effects of polymorphisms on targets.

Percent of genes containing at least one good target (blue) and percent of good targets out of possible targets as a function of DRA frequency threshold (orange). This analysis is based on N = 654 An. gambiae samples (Ag1000G data). DRA frequency threshold is the value beyond which alternative alleles are considered to be DRAs and are filtered out (i.e., a DRA frequency threshold of 0.01 means, alternative alleles with a frequency below 1% are ignored during filtering). Note the constant decline in the fraction of good targets (orange), which is not mirrored in the fraction of genes containing good targets (blue) until the DRA frequency threshold is set at <0.01. Source data are provided as a Source Data file.