2-locus TARE drives represent promising tools for effective and strongly confined population modification and are constructed with underdominance characteristics, yielding a higher introduction threshold even when drive performance is ideal.
Abstract
Gene drive systems enable rapid spread of desired transgenes throughout populations. Advances in CRISPR technology have facilitated the construction of toxin-antidote gene drives, which utilize a CRISPR nuclease as the toxin to disrupt an essential wild-type gene alongside a recoded version of the same gene as the antidote. Because these systems propagate by eliminating wild-type alleles rather than directly copying themselves like homing drives, they typically exhibit introduction thresholds, allowing them to be confined to target populations. Previous work developed the efficient Toxin-Antidote Recessive Embryo (TARE) drive, but its threshold may be too low in challenging confinement scenarios. Here, we constructed a 2-locus TARE drive system. It has underdominance characteristics, yielding a higher introduction threshold, even when drive performance is ideal. It targets the essential but haplosufficient genes hairy and sim using two different drives at different genomic locations, each targeting the gene that the other rescues. Our system involved two linked elements together with rare homology-directed repair-mediated drive conversion, reducing the threshold to compensate for fitness costs. The system showed high efficiency in individual crosses. When released into multigenerational cage populations above the introduction threshold, the drive successfully and rapidly modified the entire population, and when below this threshold, it was eliminated. Our findings indicate that 2-locus TARE drives represent promising tools for effective and strongly confined population modification.
Gene drives are potentially powerful tools, able to spread throughout target populations. They could be used to modify or suppress disease vectors, invasive species, and agricultural pests. Yet, in many scenarios, confinement of the drive to only a target population is required. Several types of drives are capable of this, among the most promising of which are CRISPR toxin-antidote drives. Though showing good performance in simple models, such drives have not been thoroughly assessed in spatially structured populations. Here we evaluate three modification drive variants with varying levels of confinement. We find that Toxin-Antidote Recessive Embryo (TARE) drive and 2-locus TARE drives can usually spread in connected populations or from a single sufficiently large release. However, they can still be stopped by a migration corridor or by a sufficiently high population density gradient. 1-locus 2-drive TARE, on the other hand, will not be able to spread outside of release areas and indeed will often retreat in the face of wild-type alleles. When these drives arrive at a point source such as a port, only the standard TARE drive has a significant chance of establishing if releases occur at sufficiently high frequency and quantity. However, in models with parental care among a limited number of offspring, TARE drives become more invasive, with lower introduction thresholds. Overall, we find that spatial and other ecological factors can substantially affect the outcome of a confined CRISPR toxin-antidote drive release.
Ziqian Xu, Yuna I. Cho, Xin-Yue Zhang et al.· bioRxiv· 0 citations
This work develops a plasmid-based reporter system in budding yeast for the rapid identification of high-performing gRNAs in budding yeast and introduces BITREx 2.0, a dual-nicking strategy that targets both sides of the gene array.
It is indicated that higher drive conversion is associated with elevated expression of the promoter-associated gene in reproductive cells, but embryo resistance allele formation correlates with excessive female germline expression, and optimal drive performance requires restricting Cas9 expression to a tight quantitative and spatiotemporal window.
Ying Wu, Yunchen Xia, Ziyin Yao et al.· Nature Communications· 0 citations
Two inducible CRISPR-based perturbation platforms are characterized in K562 human hematopoietic cells: dual-inducible CRISPRi system employing a dCas9–KRAB–DHFR–TetON architecture controlled by doxycycline and trimethoprim and single-inducible CRISPRa system employing a dCas9–VPR–TetON architecture controlled by Dox alone.
Toxin-antidote elements (TAs) are selfish DNA sequences that bias their transmission to the next generation. TAs typically consist of two linked genes: a toxin and an antidote. The toxin kills progeny that do not inherit the TA, while the antidote counteracts the toxin in progeny that inherit the TA. We previously discovered two TAs in Caenorhabditis elegans that follow the canonical TA model of two linked genes: peel-1/zeel-1 and sup-35/pha-1. Here, we report a new TA that exists in three distinct states across the C. elegans population. The canonical TA, which is found in isolates from the Hawaiian Islands, consists of two genes that encode a maternally deposited toxin (TMRL-1) and a zygotically expressed antidote (AMRL-1). The toxin induces larval lethality in embryos that do not inherit the antidote gene. A second version of the TA has lost the toxin gene but retains a partially functional antidote. Most C. elegans isolates, including the standard laboratory strain N2, carry a highly divergent allele of the toxin that has retained its activity, but have lost the antidote through pseudogenization. Multiple lines of evidence suggest that the N2 tmrl-1 allele is likely recognized by piRNAs, leading to MUT-16-dependent 22G small interfering RNA (siRNA) production and post-transcriptional silencing of the transcript. The N2 haplotype represents the first naturally occurring unlinked toxin-antidote system where the toxin is post-transcriptionally suppressed by endogenous small RNA pathways.
Stefan Zdraljevic, Laura Walter-McNeill, Giancarlo N. Bruni et al.· eLife· 0 citations
Advances in molecular biology tools are essential for streamlining and accelerating genetic engineering of cells across industrial and academic applications. While CRISPR-Cas improves genome editing efficiency, current systems have limitations and are often host specific, which restricts their versatility. This study describes a versatile CRISPR-Cas9 system for genome editing in industrially relevant Bacillus species. By adapting the well-established pJOE8999 vector-based CRISPR-Cas9 genome editing system, we constructed an inducer-independent, broad-host-range genome editing system. It maintains the benefits of low toxicity to the target cell and the cloning host as well as the ease to use of a single-plasmid CRISPR-Cas9 system. We utilized the constitutive Sigma70-type promoter from the conserved veg gene of Bacillus, to develop and test the suitability of promoter variants of different strengths for Cas9 expression. Successful gene deletions in three different Bacillus species demonstrated the versatility of the modified system for this industrially important genus. This was further confirmed by the integration of a reporter gene fusion and the introduction of a single point mutation in the genome of Bacillus licheniformis. This one-step CRISPR-based transformation protocol developed in this study enables fast genome editing workflows with minimal hands-on time. • Editing and screening of promoter variants for balanced Cas9 expression in Bacillus. • Development of a versatile inducer-independent, single-plasmid CRISPR-Cas-based system. • Verification of the modified CRISPR-based system for genome editing in different Bacilli.
Maximilian Hilkmann, Norma Welsch, M. F. Felle et al.· Applied Microbiology and Bio...· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
