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Volume 1, Issue 9

Analysis of CRISPR gene drive design in budding yeast Open Access

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 , 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 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 Cas9; 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.

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© 2019 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2019-11-01
2024-04-16
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Analysis of CRISPR gene drive design in budding yeast
Access Microbiology 1, e000059 (2019); https://doi.org/10.1099/acmi.0.000059
/content/journal/acmi/10.1099/acmi.0.000059
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