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

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.

Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes

“…bacteria, yeast, insects, and mammals (Windbichler et al 2011;Gantz and Bier 2015;DiCarlo et al 2015;Hammond et al 2016Hammond et al , 2018Champer et al 2017Champer et al , 2018KaramiNejadRanjbar et al 2018;Kyrou et al 2018;Yan and Finnigan 2018;Li et al 2019;Grunwald et al, 2019, Valderrama et al, 2019. They function by encoding the Cas9 endonuclease and an independently expressed guide RNA (gRNA) responsible for mediating DNA/RNA base pairing and cleavage at a predetermined site (Esvelt et al 2014;Bier 2015, 2016;Champer et al 2016;Marshall and Akbari 2018).…”

CRISPR-Cas System: An Approach With Potentials for COVID-19 Diagnosis and Therapeutics

“…As of now, any SARS-CoV-2 specific therapy is not available to cure COVID-19 and also, the vaccine development is estimated to take 12-18 months. Recently, CRISPR-Cas13 has emerged as a potent system having the abilityto protect host bacterial cells against bacteriophage infection by using sequence specific crRNAs (Yan and Finnigan, 2018;Yan W. X. et al, 2019). A similar strategy may also be applied to design a therapeutic agent to target the ssRNA genome of SARS-CoV-2 for managing COVID-19.…”

Advances in Molecular Tools and In Vivo Models for the Study of Human Fungal Pathogenesis

“…A fundamental design of CRISPR based gene drive employs a nuclease (Cas9 or Cas12a), guide expression cassette(s) integrated into the genome and donor ("cargo") element. The CRISPR/Cas construct is copied via DSB and homology-directed repair allowing the integration of the "cargo" element and converting cellular heterozygosity to homozygosity [39]. Such gene drives follow super-Mendelian inheritance (above 50%) allowing biased propagation of desired genetic elements within a population and eventually the entire species [38,40].…”