Genetic conversion of a split-drive into a full-drive element

Gene-drive systems offer an important new avenue for spreading beneficial traits into wild populations. Their core components, Cas9 and guide RNA (gRNA), can either be linked within a single cassette (full gene drive, fGD) or provided in two separate elements (split gene drive, sGD) wherein the gRNA-bearing element drives in the presence of an independent static source of Cas9. We previously designed a system engineered to turn split into full gene drives. Here, we provide experimental proof-of-principle for such a convertible system inserted at the spo11 locus, which is recoded to restore gene function. In multigenerational cage studies, the reconstituted spo11 fGD cassette initially drives with slower kinetics than the unlinked sGD element (using the same Mendelian vasa-Cas9 source), but eventually reaches a similar level of final introgression. Different kinetic behaviors may result from transient fitness costs associated with individuals co-inheriting Cas9 and gRNA transgenes during the drive process.

Inherently confinable split-drive systems in Drosophila

CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.

Inherently confinable split-drive systems in Drosophila

CRISPR-based gene drive systems, which copy themselves based on gene conversion mediated by the homology directed repair (HDR) pathway, have potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway that are rendered resistant to Cas9 cleavage can interrupt the spread of genedrive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences to restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles, combined with recessive Mendelian processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that were inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multi-generational cage trials, sGD follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage or lethal/sterile mosaic phenotypes, leading to inherently confineable drive outcomes.

Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

ABSTRACTCRISPR-based gene drives spread through populations bypassing the dictates of Mendelian genetics, offering a population-engineering tool for tackling vector-borne diseases, managing crop pests, and helping island conservation efforts; unfortunately, current technologies raise safety concerns for unintended gene propagation. Herein, we address this by splitting the two drive components, Cas9 and gRNAs, into separate alleles to form a novel trans-complementing split–gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This bi-component nature allows for individual transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small– molecule-controlled version to investigate the biology of component inheritance and use our system to study the maternal effects on CRISPR inheritance, impaired homology on efficiency, and resistant allele formation. Lastly, mathematical modeling of tGD spread in a population shows potential advantages for improving current gene-drive technologies for field population modification.

Using multiple reference genomes to identify and resolve annotation inconsistencies

BackgroundAdvances in sequencing technologies have led to the release of reference genomes and annotations for multiple individuals within more well-studied systems. While each of these new genome assemblies shares significant portions of synteny between each other, the annotated structure of gene models within these regions can differ. Of particular concern are split-gene misannotations, in which a single gene is incorrectly annotated as two distinct genes or two genes are incorrectly annotated as a single gene. These misannotations can have major impacts on functional prediction, estimates of expression, and many downstream analyses.ResultsWe developed a high-throughput method based on pairwise comparisons of annotations that detect potential split-gene misannotations and quantifies support for whether the genes should be merged into a single gene model. We demonstrate the utility of our method using gene annotations of three reference genomes from maize (B73, PH207, and W22), a difficult system from an annotation perspective due to the size and complexity of the genome. On average, we find several hundred of these potential split-gene misannotations in each pairwise comparison, corresponding to 3-5% of gene models across annotations. To determine which state (i.e. one gene or multiple genes) is biologically supported, we utilize RNAseq data from 10 tissues throughout development along with a novel metric and simulation framework. The methods we have developed require minimal human interaction and can be applied to future assemblies to aid in annotation efforts.ConclusionsSplit-gene misannotations occur at appreciable frequency in maize annotations. We have developed a method to easily identify and correct these misannotations. Importantly, this method is generic in that it can utilize any type of short-read expression data. Failure to account for split-gene misannotations has serious consequences for biological inference, particularly for expression-based analyses.

Development of a Confinable Gene-Drive System in the Human Disease Vector, Aedes aegypti

Aedes aegypti, the principal mosquito vector for many arboviruses that causes yellow fever, dengue, Zika, and chikungunya, increasingly infects millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has recently sparked significant enthusiasm for the genetic control of mosquito populations, however no such system has been developed in Ae. aegypti. To fill this void and demonstrate efficacy in Ae. aegypti, here we develop several CRISPR-based split-gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild Ae. aegypti populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.Significance StatementAe. aegypti is a globally distributed arbovirus vector spreading deadly pathogens to millions of people annually. Current control methods are inadequate and therefore new technologies need to be innovated and implemented. With the aim of providing new tools for controlling this pest, here we engineered and tested several split gene drives in this species. These drives functioned at very high efficiency and may provide a tool to fill the void in controlling this vector. Taken together, our results provide compelling path forward for the feasibility of future effector-linked split-drive technologies that can contribute to the safe, sustained control and potentially the elimination of pathogens transmitted by this species.