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Development of a multi-locus CRISPR gene drive system in budding yeast

Yao Yan, Gregory C. Finnigan
doi: https://doi.org/10.1101/391334
Yao Yan
1Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers, Hall, Manhattan, KS 66506 USA
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Gregory C. Finnigan
1Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers, Hall, Manhattan, KS 66506 USA
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  • For correspondence: gfinnigan@ksu.edu
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ABSTRACT

The discovery of CRISPR/Cas gene editing has allowed for major advances in many biomedical disciplines and basic research. One arrangement of this biotechnology, a nuclease-based gene drive, can rapidly deliver a genetic element through a given population and studies in fungi and metazoans have demonstrated the success of such a system. This methodology has the potential to control biological populations and contribute to eradication of insect-borne diseases, agricultural pests, and invasive species. However, there remain challenges in the design, optimization, and implementation of gene drives including concerns regarding biosafety, containment, and control/inhibition. Given the numerous gene drive arrangements possible, there is a growing need for more advanced designs. In this study, we use budding yeast to develop an artificial multi-locus gene drive system. Our minimal setup requires only a single copy of S. pyogenes Cas9 and three guide RNAs to propagate three separate gene drives. We demonstrate how this system could be used for targeted allele replacement of native genes and to suppress NHEJ repair systems by modifying DNA Ligase IV. A multi-locus gene drive configuration provides an expanded suite of options for complex attributes including pathway redundancy, combatting evolved resistance, and safeguards for control, inhibition, or reversal of drive action.

ABBREVIATIONS
CRISPR
clustered regularly interspaced short palindromic repeats;
NHEJ
non-homologous end joining;
HR
homologous recombination;
DSB
double stranded break;
sgRNA
single guide RNA fragment;
GD
CRISPR-based gene drive system;
CGM
“complete” multi-locus drive;
MGD
“minimal” multi-locus drive;
BRCT
BRCA1 C-Terminal domains;
UTR
untranslated region;
indel
insertion or deletion.
Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted August 14, 2018.
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Development of a multi-locus CRISPR gene drive system in budding yeast
Yao Yan, Gregory C. Finnigan
bioRxiv 391334; doi: https://doi.org/10.1101/391334
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Development of a multi-locus CRISPR gene drive system in budding yeast
Yao Yan, Gregory C. Finnigan
bioRxiv 391334; doi: https://doi.org/10.1101/391334

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