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.
Abstract
Gene drive can modify or suppress vector populations by spreading drive alleles. In CRISPR homing drives, regulating Cas9 expression has been effective for improving drive performance, but selecting suitable promoters is often a major challenge. Here, we evaluate 35 Cas9 constructs with distinct promoters in Drosophila melanogaster and identify associations between drive performance and single-cell RNA expression patterns of the promoter-associated genes. Our results indicate 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. For males, early germline expression produces superior performance. Thus, optimal drive performance requires restricting Cas9 expression to a tight quantitative and spatiotemporal window. Additionally, we find that an in situ construct significantly reduces potentially harmful somatic expression. Based on these results, we propose criteria for selecting promoters, providing a rationale and guidance for optimization of homing gene drives. CRISPR homing gene drives can help control disease vectors but require precise Cas9 expression. Here, the drive performance of 35 Cas9 constructs with different promoters in Drosophila were compared to single cell RNA expression patterns of promoter-associated genes.
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.
Ruobing Feng, Andrea Y.N. Tan, Zhuoran Lu et al.· bioRxiv· 1 citation
Predictable control of gene expression is essential for building genetic circuits and improving metabolic pathways, but conventional promoter libraries often behave unpredictably when genes are combined. Here we develop CRISPR-Activated Promoter-based Orthogonal expression (CAPO), a quantitative platform for controlling multiple genes in yeast. CAPO uses synthetic CRISPR-activated promoters that remain silent until matching guide RNAs recruit dCas9-VPR. We tune each gene by varying guide RNA abundance with defined T7 promoters, while keeping regulatory channels orthogonal. CAPO reaches expression levels comparable to strong native yeast promoters, maintains low background activity, and preserves promoter-strength order across different genes. We apply CAPO to program broad fluorescence color outputs and to rapidly optimize lycopene and 3-hydroxypropionic acid biosynthesis. These results establish CAPO as a scalable platform for predictable engineering of eukaryotic gene networks. Efficient bioproduction using eukaryotes, such as engineered Saccharomyces cerevisiae, requires precise control over gene expression. Here, authors develop CAPO, a CRISPR-guided system that tunes gene activity in yeast and enables multiplex colour generation and faster optimization of metabolic pathways.
Drosophila suzukii (Matsumura, 1931, Diptera: Drosophilidae) is a globally invasive pest of soft-skinned fruits that is currently controlled largely through the use of broad-spectrum insecticides. Increasing resistance to insecticides and regulatory pressures have motivated the development of genetic control strategies. We previously developed a CRISPR/Cas9-based homing gene drive targeting the coding sequence of the female-specific exon of the sex-determination gene doublesex, achieving highly efficient inheritance (94–99%) in both male and female germlines. A major limitation of homing gene drives is the formation of resistant alleles that evade cleavage yet retain gene function. Multiplexing guide RNAs (gRNAs) could reduce the formation of such functional resistance alleles. Here, we generated and tested homing constructs expressing one, two, or three gRNAs targeting different regions of the female-specific exon of doublesex, including the intron-exon splice junction. A single gRNA targeting the splice junction supported high inheritance in males but showed reduced efficiency in females. Combining this gRNA with a coding sequence-targeting guide further reduced drive efficiency, particularly in the female germline. Constructs expressing two gRNAs performed similarly whether guides were linked by transfer RNA (tRNA) sequences or expressed from independent promoters. Constructs expressing three gRNAs using tRNA processing showed consistently low drive inheritance in both sexes and low frequencies of target-site modification among non-drive progeny, consistent with reduced cleavage activity. Inheritance was significantly higher in male than female germlines for several constructs, indicating that germline context strongly influences drive performance. Our findings show that the strategy used for multi-gRNA expression, target site choice and sex-specific germline environments can influence gene drive efficiency, emphasizing the need to optimize construct design in the target species. Author Summary Spotted wing drosophila (Drosophila suzukii) is an invasive pest that damages soft skinned fruits such as berries and cherries. Control currently relies heavily on insecticides, but resistance and regulatory concerns are increasing the need for alternative approaches. We are developing genetic strategies for suppression of pest populations. We previously developed a gene editing system targeting a female-essential gene that showed very high biased inheritance (gene drive). However, a key challenge is that the system can sometimes create resistant individuals that are unaffected. One possible solution is to use using multiple gRNAs—molecules that direct gene editing to specific DNA sequences—to reduce the formation of these resistant individuals. In this study we found that using more than one gRNA reduced the efficiency of inheritance, especially in females. However, activity could be influenced by exactly where the DNA is targeted and how the guide RNAs are expressed. These results show that gene drive performance depends strongly on biological context and design choices, and that strategies must be carefully optimized in the target species.
Amarish K. Yadav, Weizhe Chen, Jackson Champer et al.· bioRxiv· 0 citations
This work cloned the promoter of the housekeeping gene eukaryotic translation elongation factor 1α (EF1Α, AAEL017096) and confirmed its transcriptional activity, and truncated the U6 promoter, expanding the genetic toolkit for Ae.
To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion increases editing efficiency by up to six-fold across diverse payloads, loci, and cell types. We test the top two factors, GJB2 and BET1L, in two distinct human models: correction of a pathogenic adenine base mutation in KCNJ13 and introduction of a cytosine base mutation in the GABAA receptor gene. Depletion of either improves base-editing outcomes by 6-fold, potentially through effects on delivery. In a patient-derived model of retinal channelopathy, knockdown of either gene improves lipid nanoparticle base editing efficiency by over 3.5-fold. This enables functional restoration of Kir7.1 ion channels in a subset of edited cells, highlighting cellular barriers as actionable targets to enhance the potency of genetic therapies. Low editing efficiency of nonviral delivery in post mitotic tissues presents a challenge to the field of gene therapy. Here, authors dissect the genetic regulators of nonviral delivery in post mitotic retinal epithelial cells describe strategies for improved base editor delivery and editing.
Shivani Saxena, Meha Kabra, Amr A. Abdeen et al.· Nature Communications· 0 citations
A novel genome-wide CRISPR screening strategy that will facilitate the systematic engineering of novel nonviral genome editing delivery methods, where the identified novel gene hits can be further used to increase editing efficiency for other therapeutically relevant cell types.
Shivani Saxena, Meha Kabra, Amr A. Abdeen et al.· bioRxiv· 2 citations