Monitoring Needs for Gene Drive Mosquito Projects: Lessons From Vector Control Field Trials and Invasive Species

As gene drive mosquito projects advance from contained laboratory testing to semi-field testing and small-scale field trials, there is a need to assess monitoring requirements to: i) assist with the effective introduction of the gene drive system at field sites, and ii) detect unintended spread of gene drive mosquitoes beyond trial sites, or resistance mechanisms and non-functional effector genes that spread within trial and intervention sites. This is of particular importance for non-localized gene drive projects, as the potential scale of intervention means that monitoring is expected to be more costly than research, development and deployment. Regarding monitoring needs for population replacement systems, lessons may be learned from experiences with Wolbachia-infected mosquitoes, and for population suppression systems, from experiences with releases of genetically sterile male mosquitoes. For population suppression systems, assessing monitoring requirements for tracking population size and detecting rare resistant alleles are priorities, while for population replacement systems, allele frequencies must be tracked, and pressing concerns include detection of gene drive alleles with non-functional effector genes, and resistance of pathogens to functional effector genes. For spread to unintended areas, open questions relate to the optimal density and placement of traps and frequency of sampling in order to detect gene drive alleles, drive-resistant alleles or non-functional effector genes while they can still be effectively managed. Invasive species management programs face similar questions, and lessons may be learned from these experiences. We explore these monitoring needs for gene drive mosquito projects progressing through the phases of pre-release, release and post-release.

Modeling CRISPR gene drives for suppression of invasive rodents using a supervised machine learning framework

Invasive rodent populations pose a threat to biodiversity across the globe. When confronted with these invaders, native species that evolved independently are often defenseless. CRISPR gene drive systems could provide a solution to this problem by spreading transgenes among invaders that induce population collapse, and could be deployed even where traditional control methods are impractical or prohibitively expensive. Here, we develop a high-fidelity model of an island population of invasive rodents that includes three types of suppression gene drive systems. The individual-based model is spatially explicit, allows for overlapping generations and a fluctuating population size, and includes variables for drive fitness, efficiency, resistance allele formation rate, as well as a variety of ecological parameters. The computational burden of evaluating a model with such a high number of parameters presents a substantial barrier to a comprehensive understanding of its outcome space. We therefore accompany our population model with a meta-model that utilizes supervised machine learning to approximate the outcome space of the underlying model with a high degree of accuracy. This enables us to conduct an exhaustive inquiry of the population model, including variance-based sensitivity analyses using tens of millions of evaluations. Our results suggest that sufficiently capable gene drive systems have the potential to eliminate island populations of rodents under a wide range of demographic assumptions, though only if resistance can be kept to a minimal level. This study highlights the power of supervised machine learning to identify the key parameters and processes that determine the population dynamics of a complex evolutionary system.

Weakly deleterious natural genetic variation amplifies probability of resistance in multiplexed gene drive systems

Evolution of resistance is a major barrier to successful deployment of gene drive systems to suppress natural populations. Multiplexed guide RNAs that require resistance mutations in all target cut sites is a promising strategy to overcome resistance. Using novel stochastic simulations that accurately model evolution at very large population sizes, we explore the probability of resistance due to three important mechanisms: 1) non-homologous end-joining mutations, 2) single nucleotide mutants arising de novo or, 3) single nucleotide polymorphisms pre-existing as standing variation. If the fraction of functional end-joining mutants is rare, we show that standing variation dominates, via a qualitatively new phenomenon where weakly deleterious variants significantly amplify the probability of multi-site resistance. This means resistance can be probable even with many target sites in not very large populations. This result has broad application to resistance arising in multi-site evolutionary scenarios including the evolution of vaccine escape mutations in large populations.

Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline

Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a “gene drive,” to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an “insertion/deletion” (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of “super-mendelian” inheritance from both male and female mice.

Analysis of a Cas12a-based gene-drive system in budding yeast

The discovery and adaptation of CRISPR/Cas systems within molecular biology has provided advances across biological research, agriculture and human health. Genomic manipulation through use of a CRISPR nuclease and programmed guide RNAs has become a common and widely accessible practice. The identification and introduction of new engineered variants and orthologues of Cas9 as well as alternative CRISPR systems such as the type V group have provided additional molecular options for editing. These include distinct PAM requirements, staggered DNA double-strand break formation, and the ability to multiplex guide RNAs from a single expression construct. Use of CRISPR/Cas has allowed for the construction and testing of a powerful genetic architecture known as a gene drive within eukaryotic model systems. Our previous work developed a drive within budding yeast using Streptococcus pyogenes Cas9. Here, we installed the type V Francisella novicida Cas12a (Cpf1) nuclease gene and its corresponding guide RNA to power a highly efficient artificial gene drive in diploid yeast. We examined the consequence of altering guide length or introduction of individual mutational substitutions to the crRNA sequence. Cas12a-dependent gene-drive function required a guide RNA of at least 18 bp and could not tolerate most changes within the 5′ end of the crRNA.