Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae

Abstract

BackgroundThe bacterial CRISPR/Cas genome editing system has provided a major breakthrough in molecular biology. One use of this technology is within a nuclease-based gene drive. This type of system can install a genetic element within a population at unnatural rates. Combatting of vector-borne diseases carried by metazoans could benefit from a delivery system that bypasses traditional Mendelian laws of segregation. Recently, laboratory studies in fungi, insects, and even mice, have demonstrated successful propagation of CRISPR gene drives and the potential utility of this type of mechanism. However, current gene drives still face challenges including evolved resistance, containment, and the consequences of application in wild populations. Additional research into molecular mechanisms that would allow for control, titration, and inhibition of drive systems is needed.ResultsIn this study, we use artificial gene drives in budding yeast to explore mechanisms to modulate nuclease activity of Cas9 through its nucleocytoplasmic localization. We examine non-native nuclear localization sequences (both NLS and NES) on Cas9 fusion proteins in vivo through fluorescence microscopy and genomic editing. Our results demonstrate that mutational substitutions to nuclear signals and combinatorial fusions can both modulate the level of gene drive activity within a population of cells.ConclusionsThese findings have implications for control of traditional nuclease-dependent editing and use of gene drive systems within other organisms. For instance, initiation of a nuclear export mechanism to Cas9 could serve as a molecular safeguard within an active gene drive to reduce or eliminate editing.Electronic supplementary materialThe online version of this article (10.1186/s40694-019-0065-x) contains supplementary material, which is available to authorized users.

Analysis of a Cas12a-based gene-drive system in budding yeast

“…Our previous work in yeast has employed use of the type II S. pyogenes Cas9 to power gene-drive systems within diploid cells [24–26, 28]. Given the discovery and widespread availability of other CRISPR nucleases and engineered variants, we predicted that use of alternative systems may also allow for successful gene-drive (GD) activity in vivo .…”

“…To date, these systems have been developed and studied in fungi, metazoans, bacteria and viruses under laboratory conditions [18, 19, 21–23]. Our previous work has developed a safe and ‘artificial’ drive system in budding yeast for study of various aspects of CRISPR/Cas editing such as nucleocytoplasmic trafficking, guide RNA specificity and anti-CRISPR inhibition [24–26].…”

The Challenges in Developing Efficient and Robust Synthetic Homing Endonuclease Gene Drives

“…The future design of HEGs may be aided by studying the effect of “stacking” multiple limited regulatory mechanisms. In addition to the use of promoter/5′UTR and 3′UTR sequences, other endogenous regulation mechanisms can be included, such as tissue-specific splicing ( Salles et al, 2002 ; Tsujimoto et al, 2013 ; Sutton et al, 2016 ), modulation of protein degradation ( Chassin et al, 2019 ), sub-cellular localisation ( Goeckel et al, 2019 ), and inclusion of miRNA binding sites ( Loya et al, 2009 ). Ideally each regulatory system should make as limited and well defined a change as possible.…”

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

“…Subsequently, Halder and colleagues developed an optimized protocol for CRISPR based gene-drive system to study genetic interactions in C. albicans [45]. Furthermore, improvements in CRISPR-Cas9 gene drives have been developed providing means to markerless-selection, bias towards homology-directed repair, increased layers of biosafety, maximizing potential redundancy and regulating gene-drive activity [39,41,42,46].…”