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 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.
Highlights
The discovery and implementation of the clustered regularly interspaced short palindromic repeat (CRISPR) gene editing system has revolutionized countless fields and sub-specialties across molecular biology and biotechnology to improve human health, agriculture, ecological control, and beyond
The proposal to increase the number of targeted double strand breaks by the single nuclease of choice (e.g. S. pyogenes Cas9) would greatly aid in combatting resistance[35,36,37]
We have developed a multi-locus CRISPR gene drive with a minimal design (MGD) that allows for multiplexing of Cas[9] in trans across three distinct chromosomal locations (Fig. 1)
Summary
The discovery and implementation of the clustered regularly interspaced short palindromic repeat (CRISPR) gene editing system has revolutionized countless fields and sub-specialties across molecular biology and biotechnology to improve human health, agriculture, ecological control, and beyond. Creation of a DSB followed by HR-based repair (using the gene drive-containing DNA as a donor) causes the entire artificial construct (Cas[9], the sgRNA, and any desired “cargo”) to be copied; in this way, a heterozygous cell is automatically converted to the homozygous state. This super-Mendelian genetic arrangement allows for the forced propagation of a genetic element within a population and has the potential to modify entire species on a global scale[8,9]. Our method includes multiple layers of genetic safeguards as well as recommendations for future designs of multi-locus drive systems
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