Analysis of CRISPR gene drive design in budding yeast.

Autor:	Yan Y; Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506, USA., Finnigan GC; Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506, USA.
Jazyk:	angličtina
Zdroj:	Access microbiology [Access Microbiol] 2019 Sep 11; Vol. 1 (9), pp. e000059. Date of Electronic Publication: 2019 Sep 11 (Print Publication: 2019).
DOI:	10.1099/acmi.0.000059
Abstrakt:	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. Competing Interests: G. C. F. (Kansas State University) filed for a provisional patent on 29 September 2017 (US Provisional Patent Application Serial No. 62/565,651, ‘Programmed modulation of CRISPR/Cas9 activity’) followed by patent filing on 31 January 2018 regarding some of the data presented in this work. A patent was filed (G. C. F.) on 20 April 2017 by the University of California, Berkeley, ‘Methods and Compositions for Genomic Editing’ International Application No. PCT/US2017/028676 and published as No. WO 2017/189336 A1 on 2 November 2017, for the artificial target sites (u1′/u2/u2′) used in this study. G. C. F. declares no non-financial conflict of interest. Y. Y. declares no conflict of interest of any kind. (© 2019 The Authors.)
Databáze:	MEDLINE
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