Dataset Information

Analysis of CRISPR gene drive design in budding yeast.

ABSTRACT: Control of biological populations remains a critical goal to address the challenges facing ecosystems and agriculture and those posed by human disease, including pests, parasites, pathogens and invasive species. A particular architecture of the CRISPR/Cas biotechnology - a gene drive - has the potential to modify or eliminate populations on a massive scale. Super-Mendelian inheritance has now been demonstrated in both fungi and metazoans, including disease vectors such as mosquitoes. Studies in yeast and fly model systems have developed a number of molecular safeguards to increase biosafety and control over drive systems in vivo, including titration of nuclease activity, anti-CRISPR-dependent inhibition and use of non-native DNA target sites. We have developed a CRISPR/Cas9 gene drive in Saccharomyces cerevisiae that allows for the safe and rapid examination of alternative drive designs and control mechanisms. In this study, we tested whether non-homologous end-joining (NHEJ) had occurred within diploid cells displaying a loss of the target allele following drive activation and did not detect any instances of NHEJ within multiple sampled populations. We also demonstrated successful multiplexing using two additional non-native target sequences. Furthermore, we extended our analysis of 'resistant' clones that still harboured both the drive and target selection markers following expression of Streptococcus pyogenes Cas9; de novo mutation or NHEJ-based repair could not explain the majority of these heterozygous clones. Finally, we developed a second-generation gene drive in yeast with a guide RNA cassette integrated within the drive locus with a near 100 % success rate; resistant clones in this system could also be reactivated during a second round of Cas9 induction.

SUBMITTER: Yan Y

PROVIDER: S-EPMC7472540 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Analysis of CRISPR gene drive design in budding yeast.

Yan Yao Y Finnigan Gregory C GC

Access microbiology 20190911 9

Control of biological populations remains a critical goal to address the challenges facing ecosystems and agriculture and those posed by human disease, including pests, parasites, pathogens and invasive species. A particular architecture of the CRISPR/Cas biotechnology - a gene drive - has the potential to modify or eliminate populations on a massive scale. Super-Mendelian inheritance has now been demonstrated in both fungi and metazoans, including disease vectors such as mosquitoes. Studies in ...[more]

PMID: 32974560

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Project description:BACKGROUND:CRISPR gene drive systems allow the rapid spread of a genetic construct throughout a population. Such systems promise novel strategies for the management of vector-borne diseases and invasive species by suppressing a target population or modifying it with a desired trait. However, current homing-type drives have two potential shortcomings. First, they can be thwarted by the rapid evolution of resistance. Second, they lack any mechanism for confinement to a specific target population. In this study, we conduct a comprehensive performance assessment of several new types of CRISPR-based gene drive systems employing toxin-antidote (TA) principles, which should be less prone to resistance and allow for the confinement of drives to a target population due to invasion frequency thresholds. RESULTS:The underlying principle of the proposed CRISPR toxin-antidote gene drives is to disrupt an essential target gene while also providing rescue by a recoded version of the target as part of the drive allele. Thus, drive alleles tend to remain viable, while wild-type targets are disrupted and often rendered nonviable, thereby increasing the relative frequency of the drive allele. Using individual-based simulations, we show that Toxin-Antidote Recessive Embryo (TARE) drives targeting an haplosufficient but essential gene (lethal when both copies are disrupted) can enable the design of robust, regionally confined population modification strategies with high flexibility in choosing promoters and targets. Toxin-Antidote Dominant Embryo (TADE) drives require a haplolethal target gene and a germline-restricted promoter, but they could permit faster regional population modification and even regionally confined population suppression. Toxin-Antidote Dominant Sperm (TADS) drives can be used for population modification or suppression. These drives are expected to spread rapidly and could employ a variety of promoters, but unlike TARE and TADE, they would not be regionally confined and also require highly specific target genes. CONCLUSIONS:Overall, our results suggest that CRISPR-based TA gene drives provide promising candidates for flexible ecological engineering strategies in a variety of organisms.

Dataset Information

Analysis of CRISPR gene drive design in budding yeast.

Publications

Analysis of CRISPR gene drive design in budding yeast.

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