CRISPR/Cas9 manipulations are possible in many insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of CRISPR-based (clustered regularly interspaced short palindromic repeats) genome-editing tools and methodologies are dependent upon direct experimentation. One useful technique is Cas9-dependent homologous recombination, which is a critical tool for studying gene function but also for developing pest related applications like gene drive. Here, we report our attempts to induce Cas9 homology directed repair (HDR) and subsequent gene drive in Tribolium castaneum (Herbst; Insecta: Coleoptera: Tenebrionidae). Utilizing constructs containing 1 or 2 target gRNAs in combination with Cas9 under 2 different promoters and corresponding homology arms, we found a high incidence of CRISPR/Cas9 induced mutations but no evidence of homologous recombination. Even though the generated constructs provide new resources for CRISPR/Cas9 modification of the Tribolium genome, our results suggest that additional modifications and increased sample sizes will be necessary to increase the potential and detection for HDR of the Tribolium genome.

CRISPR-Cas9 homing gene drives are designed to induce a targeted double-stranded DNA break at a wild type allele (\'recipient\'), which, when repaired by the host cell, is converted to the drive allele from the homologous (\'donor\') chromosome. Germline localisation of this process leads to super-Mendelian inheritance of the drive and the rapid spread of linked traits, offering a novel strategy for population control through the deliberate release of drive individuals. During the homology-based DNA repair, additional segments of the recipient chromosome may convert to match the donor, potentially impacting carrier fitness and strategy success. Using Anopheles gambiae strains with variations around the drive target site, here we assess the extent and nature of chromosomal conversion. We show both homing and meiotic drive contribute as mechanisms of inheritance bias. Additionally, over 80% of homing events resolve within 50 bp of the chromosomal break, enabling rapid gene drive transfer into locally-adapted genetic backgrounds.

Gene drive technology has been recognized for its potential to provide durable and cost-effective solutions for previously intractable problems in public health, conservation, and agriculture. In recognition of the rapid advances in this field, in 2016 the U.S. National Academies of Sciences, Engineering, and Medicine issued a report making several recommendations aimed at researchers, funders, and policymakers for the safe and responsible research and development of gene drive technology. Subsequently, in 2017 sixteen global organizations self-identifying as sponsors and supporters of gene drive research became public signatories committed to the \'Principles for Gene Drive Research\' which were inspired by the report\'s recommendations. Herein we reflect on the progress of gene drive research in relation to the ethical principles laid out and committed to by the signatories to the Principles. Our analysis indicates high levels of alignment with the Principles in the field of gene drive research. The manuscript also discusses the Gene Drive Research Forum, which had its genesis in the publication of the Principles. Discussions between participants at the latest meeting of the Forum point to the work that lies ahead for gene drive research in line with the Principles. Going forward the gene drive research community can productively focus on: i) safety and efficacy criteria for open release, ii) risk assessment frameworks and methods, iii) more downstream technical, regulatory and policy considerations for field evaluations and implementation, iv) continued transparency and developing mechanisms of accountability, and v) strengthening capacity in locales of potential release and expected drive spread.

First synthetic \"gene drive\" for plants could help tame weeds-or transform them.

Elimination of malaria has become a United Nations member states target: Target 3.3 of the sustainable development goal no. 3 (SDG3). Despite the measures taken, the attainment of this goal is jeopardized by an alarming trend of increasing malaria case incidence. Globally, there were an estimated 241 million malaria cases in 2020 in 85 malaria-endemic countries, increasing from 227 million in 2019. Malaria case incidence was 59, which means effectively no changes in the numbers occurred, compared with the baseline 2015. Jennifer Doudna-co-inventor of CRISPR/Cas9 technology-claims that CRISPR holds the potential to lessen or even eradicate problems lying in the centre of SDGs. On the same note, CRISPR/Cas9-mediated mosquito-targeting gene drives (MGD) are perceived as a potential means to turn this trend back and put momentum into the malaria elimination effort. This paper assessed two of the critical elements of the World Health Organization Genetically modified mosquitoes (WHO GMM) Critical Pathway framework: the community and stakeholders\' engagement (inability to employ widely used frameworks, segmentation of the public, \'bystander\' status, and guidelines operationalization) and the regulatory landscape (lex generali, \'goldilocks dilemma\', and mode of regulation) concerning mosquito-oriented gene drives (MGD) advances. Based on the assessment findings, the author believes that CRISPR/Cas-9-mediated MGD will not contribute to the attainment of SDG3 (Target 3.3), despite the undisputable technology\'s potential. This research pertains to the state of knowledge, legal frameworks, and legislature, as of November 2022.

Gene drive alleles that can bias their own inheritance could engineer populations for control of disease vectors, invasive species and agricultural pests. There are successful examples of suppression drives and confined modification drives, but developing confined suppression drives has proven more difficult. However, CRISPR-based toxin-antidote dominant embryo (TADE) suppression drive may fill this niche. It works by targeting and disrupting a haplolethal target gene in the germline with its gRNAs while rescuing this target. It also disrupts a female fertility gene by driving insertion or additional gRNAs. Here, we used a reaction-diffusion model to assess drive performance in continuous space, where outcomes can be substantially different from those in panmictic populations. We measured drive wave speed and found that moderate fitness costs or target gene disruption in the early embryo from maternally deposited nuclease can eliminate the drive\'s ability to form a wave of advance. We assessed the required release size, and finally we investigated migration corridor scenarios. It is often possible for the drive to suppress one population and then persist in the corridor without invading the second population, a potentially desirable outcome. Thus, even imperfect variants of TADE suppression drive may be excellent candidates for confined population suppression.

Engagement is an important component in the advancement of gene-drive vector control research programs as developers look to transition the technology from the laboratory to the field. As research advances and engagement surrounding this novel technology is put into practice, knowledge can be gained from practical experiences and applications in the field. A relationship-based model (RBM) provides a framework for end-user development of engagement programs and strategies. The model places end users at the center of the engagement decision-making processes rather than as recipients of predetermined strategies, methods, and definitions. Successful RBM application for healthcare delivery has previously been demonstrated, and the University of California Malaria Initiative (UCMI) has applied this model to its gene-drive program in the Democratic Republic of São Tomé and Príncipe. The model emphasizes the importance of local leadership in the planning, development, and implementation of all phases of project engagement. The primary aim of this paper is to translate the model from paper to practice and provide a transparent description, using practical examples, of the UCMI program implementation of RBM at its field site. End-user development of the UCMI engagement program provides a unique approach to the development of ethical, transparent, and effective engagement strategies for malaria control programs. This paper may also serve as a reference and example for projects looking to establish an engagement program model that integrates end-user groups in the decision-making processes surrounding engagement.

Gene drive elements promote the spread of linked traits and can be used to change the composition or fate of wild populations. Cleave and Rescue (ClvR) drive elements sit at a fixed chromosomal position and include a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene and a recoded version of the essential gene resistant to cleavage. ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. Here we demonstrate the essential features of the ClvR gene drive in the plant Arabidopsis thaliana through killing of gametes that fail to inherit a ClvR that targets the essential gene YKT61. Resistant alleles, which can slow or prevent drive, were not observed. Modelling shows plant ClvRs are robust to certain failure modes and can be used to rapidly drive population modification or suppression. Possible applications are discussed.

Engineered sex ratio distorters (SRDs) have been proposed as a powerful component of genetic control strategies designed to suppress harmful insect pests. Two types of CRISPR-based SRD mechanisms have been proposed: X-shredding, which eliminates X-bearing sperm, and X-poisoning, which eliminates females inheriting disrupted X-chromosomes. These differences can have a profound impact on the population dynamics of SRDs when linked to the Y-chromosome: an X-shredder is invasive, constituting a classical meiotic Y-drive, whereas X-poisoning is self-limiting, unable to invade but also insulated from selection. Here, we establish X-poisoning strains in the malaria vector Anopheles gambiae targeting three X-linked genes during spermatogenesis, resulting in male bias. We find that sex distortion is primarily driven by a loss of X-bearing sperm, with limited evidence for postzygotic lethality of female progeny. By leveraging a Drosophila melanogaster model, we show unambiguously that engineered SRD traits can operate differently in these two insects. Unlike X-shredding, X-poisoning could theoretically operate at early stages of spermatogenesis. We therefore explore premeiotic Cas9 expression to target the mosquito X-chromosome. We find that, by pre-empting the onset of meiotic sex chromosome inactivation, this approach may enable the development of Y-linked SRDs if mutagenesis of spermatogenesis-essential genes is functionally balanced.

Gene drive systems could be a viable strategy to prevent pathogen transmission or suppress vector populations by propagating drive alleles with super-Mendelian inheritance. CRISPR-based homing gene drives convert wild type alleles into drive alleles in heterozygotes with Cas9 and gRNA. It is thus desirable to identify Cas9 promoters that yield high drive conversion rates, minimize the formation rate of resistance alleles in both the germline and the early embryo, and limit somatic Cas9 expression. In Drosophila, the nanos promoter avoids leaky somatic expression, but at the cost of high embryo resistance from maternally deposited Cas9. To improve drive efficiency, we test eleven Drosophila melanogaster germline promoters. Some achieve higher drive conversion efficiency with minimal embryo resistance, but none completely avoid somatic expression. However, such somatic expression often does not carry detectable fitness costs for a rescue homing drive targeting a haplolethal gene, suggesting somatic drive conversion. Supporting a 4-gRNA suppression drive, one promoter leads to a low drive equilibrium frequency due to fitness costs from somatic expression, but the other outperforms nanos, resulting in successful suppression of the cage population. Overall, these Cas9 promoters hold advantages for homing drives in Drosophila species and may possess valuable homologs in other organisms.