Gene drive designs for efficient and localisable population suppression using Y-linked editors.

The sterile insect technique (SIT) has been successful in controlling some pest species but is not practicable for many others due to the large number of individuals that need to be reared and released. Previous computer modelling has demonstrated that the release of males carrying a Y-linked editor that kills or sterilises female descendants could be orders of magnitude more efficient than SIT while still remaining spatially restricted, particularly if combined with an autosomal sex distorter. In principle, further gains in efficiency could be achieved by using a self-propagating double drive design, in which each of the two components (the Y-linked editor and the sex ratio distorter) boosted the transmission of the other. To better understand the expected dynamics and impact of releasing constructs of this new design we have analysed a deterministic population genetic and population dynamic model. Our modelling demonstrates that this design can suppress a population from very low release rates, with no invasion threshold. Importantly, the design can work even if homing rates are low and sex chromosomes are silenced at meiosis, potentially expanding the range of species amenable to such control. Moreover, the predicted dynamics and impacts can be exquisitely sensitive to relatively small (e.g., 25%) changes in allele frequencies in the target population, which could be exploited for sequence-based population targeting. Analysis of published Anopheles gambiae genome sequences indicates that even for weakly differentiated populations with an FST of 0.02 there may be thousands of suitably differentiated genomic sites that could be used to restrict the spread and impact of a release. Our proposed design, which extends an already promising development pathway based on Y-linked editors, is therefore a potentially useful addition to the menu of options for genetic biocontrol.

Driving down malaria transmission with engineered gene drives.

The last century has witnessed the introduction, establishment and expansion of mosquito-borne diseases into diverse new geographic ranges. Malaria is transmitted by female Anopheles mosquitoes. Despite making great strides over the past few decades in reducing the burden of malaria, transmission is now on the rise again, in part owing to the emergence of mosquito resistance to insecticides, antimalarial drug resistance and, more recently, the challenges of the COVID-19 pandemic, which resulted in the reduced implementation efficiency of various control programs. The utility of genetically engineered gene drive mosquitoes as tools to decrease the burden of malaria by controlling the disease-transmitting mosquitoes is being evaluated. To date, there has been remarkable progress in the development of CRISPR/Cas9-based homing endonuclease designs in malaria mosquitoes due to successful proof-of-principle and multigenerational experiments. In this review, we examine the lessons learnt from the development of current CRISPR/Cas9-based homing endonuclease gene drives, providing a framework for the development of gene drive systems for the targeted control of wild malaria-transmitting mosquito populations that overcome challenges such as with evolving drive-resistance. We also discuss the additional substantial works required to progress the development of gene drive systems from scientific discovery to further study and subsequent field application in endemic settings.

Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying ('homing') therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.

Unravelling population structure heterogeneity within the genome of the malaria vector Anopheles gambiae.

BACKGROUND: Whole genome re-sequencing provides powerful data for population genomic studies, allowing robust inferences of population structure, gene flow and evolutionary history. For the major malaria vector in Africa, Anopheles gambiae, other genetic aspects such as selection and adaptation are also important. In the present study, we explore population genetic variation from genome-wide sequencing of 765 An. gambiae and An. coluzzii specimens collected from across Africa. We used t-SNE, a recently popularized dimensionality reduction method, to create a 2D-map of An. gambiae and An. coluzzii genes that reflect their population structure similarities. RESULTS: The map allows intuitive navigation among genes distributed throughout the so-called "mainland" and numerous surrounding "island-like" gene clusters. These gene clusters of various sizes correspond predominantly to low recombination genomic regions such as inversions and centromeres, and also to recent selective sweeps. Because this mosquito species complex has been studied extensively, we were able to support our interpretations with previously published findings. Several novel observations and hypotheses are also made, including selective sweeps and a multi-locus selection event in Guinea-Bissau, a known intense hybridization zone between An. gambiae and An. coluzzii. CONCLUSIONS: Our results present a rich dataset that could be utilized in functional investigations aiming to shed light onto An. gambiae s.l genome evolution and eventual speciation. In addition, the methodology presented here can be used to further characterize other species not so well studied as An. gambiae, shortening the time required to progress from field sampling to the identification of genes and genomic regions under unique evolutionary processes.

Double drives and private alleles for localised population genetic control.

Synthetic gene drive constructs could, in principle, provide the basis for highly efficient interventions to control disease vectors and other pest species. This efficiency derives in part from leveraging natural processes of dispersal and gene flow to spread the construct and its impacts from one population to another. However, sometimes (for example, with invasive species) only specific populations are in need of control, and impacts on non-target populations would be undesirable. Many gene drive designs use nucleases that recognise and cleave specific genomic sequences, and one way to restrict their spread would be to exploit sequence differences between target and non-target populations. In this paper we propose and model a series of low threshold double drive designs for population suppression, each consisting of two constructs, one imposing a reproductive load on the population and the other inserted into a differentiated locus and controlling the drive of the first. Simple deterministic, discrete-generation computer simulations are used to assess the alternative designs. We find that the simplest double drive designs are significantly more robust to pre-existing cleavage resistance at the differentiated locus than single drive designs, and that more complex designs incorporating sex ratio distortion can be more efficient still, even allowing for successful control when the differentiated locus is neutral and there is up to 50% pre-existing resistance in the target population. Similar designs can also be used for population replacement, with similar benefits. A population genomic analysis of CRISPR PAM sites in island and mainland populations of the malaria mosquito Anopheles gambiae indicates that the differentiation needed for our methods to work can exist in nature. Double drives should be considered when efficient but localised population genetic control is needed and there is some genetic differentiation between target and non-target populations.

A novel hydrogen peroxide evolved CHO host can improve the expression of difficult to express bispecific antibodies.

The manufacture of bispecific antibodies by Chinese hamster ovary (CHO) cells is often hindered by lower product yields compared to monoclonal antibodies. Recently, reactive oxygen species have been shown to negatively impact antibody production. By contrast, strategies to boost cellular antioxidant capacity appear to be beneficial for recombinant protein expression. With this in mind, we generated a novel hydrogen peroxide evolved host using directed host cell evolution. Here we demonstrate that this host has heritable resistance to hydrogen peroxide over many generations, displays enhanced antioxidant capacity through the upregulation of several, diverse antioxidant defense genes such as those involved in glutathione synthesis and turnover, and has improved glutathione content. Additionally, we show that this host has significantly improved transfection recovery times, improved growth and viability properties in a fed-batch production process, and elevated expression of two industrially relevant difficult to express bispecific antibodies compared to unevolved CHO control host cells. These findings demonstrate that host cell evolution represents a powerful methodology for improving specific host cell characteristics that can positively impact the expression of difficult to express biotherapeutics.

Multiple Alternative Promoters and Alternative Splicing Enable Universal Transcription-Based Logic Computation in Mammalian Cells.

Multi-input logic gene circuits can enable sophisticated control of cell function, yet large-scale synthetic circuitry in mammalian cells has relied on post-transcriptional regulation or recombinase-triggered state transitions. Large-scale transcriptional logic, on the other hand, has been challenging to implement. Inspired by a naturally found regulatory strategy of using multiple alternative promoters, followed by alternative splicing, we developed a scalable and compact platform for transcriptional OR logic using inputs to those promoters. The platform is extended to implement disjunctive normal form (DNF) computations capable of implementing arbitrary logic rules. Specifically, AND logic is implemented at individual promoters using synergistic transcriptional inputs, and NOT logic via microRNA inputs targeting unique exon sequences driven by those promoters. Together, these regulatory programs result in DNF-like logic control of output gene expression. The approach offers flexibility for building complex logic programs in mammalian cells.

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex.

A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.

A circuit for detection of interaural time differences in the nucleus laminaris of turtles.

The physiological hearing range of turtles is approximately 50-1000 Hz, as determined by cochlear microphonics ( Wever and Vernon, 1956a). These low frequencies can constrain sound localization, particularly in red-eared slider turtles, which are freshwater turtles with small heads and isolated middle ears. To determine if these turtles were sensitive to interaural time differences (ITDs), we investigated the connections and physiology of their auditory brainstem nuclei. Tract tracing experiments showed that cranial nerve VIII bifurcated to terminate in the first-order nucleus magnocellularis (NM) and nucleus angularis (NA), and the NM projected bilaterally to the nucleus laminaris (NL). As the NL received inputs from each side, we developed an isolated head preparation to examine responses to binaural auditory stimulation. Magnocellularis and laminaris units responded to frequencies from 100 to 600 Hz, and phase-locked reliably to the auditory stimulus. Responses from the NL were binaural, and sensitive to ITD. Measures of characteristic delay revealed best ITDs around ±200 µs, and NL neurons typically had characteristic phases close to 0, consistent with binaural excitation. Thus, turtles encode ITDs within their physiological range, and their auditory brainstem nuclei have similar connections and cell types to other reptiles.

Underwater Hearing in Turtles.

The hearing of turtles is poorly understood compared with the other reptiles. Although the mechanism of transduction of sound into a neural signal via hair cells has been described in detail, the rest of the auditory system is largely a black box. What is known is that turtles have higher hearing thresholds than other reptiles, with best frequencies around 500 Hz. They also have lower underwater hearing thresholds than those in air, owing to resonance of the middle ear cavity. Further studies demonstrated that all families of turtles and tortoises share a common middle ear cavity morphology, with scaling best suited to underwater hearing. This supports an aquatic origin of the group. Because turtles hear best under water, it is important to examine their vulnerability to anthropogenic noise. However, the lack of basic data makes such experiments difficult because only a few species of turtles have published audiograms. There are also almost no behavioral data available (understandable due to training difficulties). Finally, few studies show what kinds of sounds are behaviorally relevant. One notable paper revealed that the Australian snake-necked turtle (Chelodina oblonga) has a vocal repertoire in air, at the interface, and under water. Findings like these suggest that there is more to the turtle aquatic auditory scene than previously thought.

Middle ear cavity morphology is consistent with an aquatic origin for testudines.

The position of testudines in vertebrate phylogeny is being re-evaluated. At present, testudine morphological and molecular data conflict when reconstructing phylogenetic relationships. Complicating matters, the ecological niche of stem testudines is ambiguous. To understand how turtles have evolved to hear in different environments, we examined middle ear morphology and scaling in most extant families, as well as some extinct species, using 3-dimensional reconstructions from micro magnetic resonance (MR) and submillimeter computed tomography (CT) scans. All families of testudines exhibited a similar shape of the bony structure of the middle ear cavity, with the tympanic disk located on the rostrolateral edge of the cavity. Sea Turtles have additional soft tissue that fills the middle ear cavity to varying degrees. When the middle ear cavity is modeled as an air-filled sphere of the same volume resonating in an underwater sound field, the calculated resonances for the volumes of the middle ear cavities largely fell within testudine hearing ranges. Although there were some differences in morphology, there were no statistically significant differences in the scaling of the volume of the bony middle ear cavity with head size among groups when categorized by phylogeny and ecology. Because the cavity is predicted to resonate underwater within the testudine hearing range, the data support the hypothesis of an aquatic origin for testudines, and function of the middle ear cavity in underwater sound detection.

Distributions of active spinal cord neurons during swimming and scratching motor patterns.

The spinal cord can generate motor patterns underlying several kinds of limb movements. Many spinal interneurons are multifunctional, contributing to multiple limb movements, but others are specialized. It is unclear whether anatomical distributions of activated neurons differ for different limb movements. We examined distributions of activated neurons for locomotion and scratching using an activity-dependent dye. Adult turtles were stimulated to generate repeatedly forward swimming, rostral scratching, pocket scratching, or caudal scratching motor patterns, while sulforhodamine 101 was applied to the spinal cord. Sulforhodamine-labeled neurons were widely distributed rostrocaudally, dorsoventrally, and mediolaterally after each motor pattern, concentrated bilaterally in the deep dorsal horn, the lateral intermediate zone, and the dorsal to middle ventral horn. Labeled neurons were common in all hindlimb enlargement segments and the pre-enlargement segment following swimming and scratching, but a significantly higher percentage were in the rostral segments following swimming than rostral scratching. These findings suggest that largely the same spinal regions are activated during swimming and scratching, but there are some differences that may indicate locations of behaviorally specialized neurons. Finally, the substantial inter-animal variability following a single kind of motor pattern may indicate that essentially the same motor output is generated by anatomically variable networks.

Specialization for underwater hearing by the tympanic middle ear of the turtle, Trachemys scripta elegans.

Turtles, like other amphibious animals, face a trade-off between terrestrial and aquatic hearing. We used laser vibrometry and auditory brainstem responses to measure their sensitivity to vibration stimuli and to airborne versus underwater sound. Turtles are most sensitive to sound underwater, and their sensitivity depends on the large middle ear, which has a compliant tympanic disc attached to the columella. Behind the disc, the middle ear is a large air-filled cavity with a volume of approximately 0.5 ml and a resonance frequency of approximately 500 Hz underwater. Laser vibrometry measurements underwater showed peak vibrations at 500-600 Hz with a maximum of 300 µm s(-1) Pa(-1), approximately 100 times more than the surrounding water. In air, the auditory brainstem response audiogram showed a best sensitivity to sound of 300-500 Hz. Audiograms before and after removing the skin covering reveal that the cartilaginous tympanic disc shows unchanged sensitivity, indicating that the tympanic disc, and not the overlying skin, is the key sound receiver. If air and water thresholds are compared in terms of sound intensity, thresholds in water are approximately 20-30 dB lower than in air. Therefore, this tympanic ear is specialized for underwater hearing, most probably because sound-induced pulsations of the air in the middle ear cavity drive the tympanic disc.