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

Abstract

The 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 vectorborne 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. In this study, we use an artificial gene drive system in budding yeast to explore mechanisms to modulate nuclease activity of Cas9 through its nucleocytoplasmic localization. We examine non-native nuclear localization sequences on Cas9 fusion proteins in vivo and demonstrate that appended signals can titrate gene drive activity and serve as a potential molecular safeguard.

Reflection on the Challenges, Accomplishments, and New Frontiers of Gene Drives

“…In this regard, Roggenkamp et al [109] reported the drive activity to be contingent on four conserved features: the Cas9 protein level, the sgRNA uniqueness, the Cas9 nucleocytoplasmic shuttling, and lastly, the novel Cas9-Cas9 tandem fusions. In their latest study, Goeckel et al [110] expanded upon their previous work to assess the ability to modulate the nuclease activity of Cas9 in S. cerevisiae based on its nucleocytoplasmic localization. Authors examined nonnative nuclear localization sequences (both nuclear localization signal (NLS) and BioDesign Research nuclear export signal (NES)) on Cas9 fusion proteins and demonstrated that mutational substitutions to nuclear signals and combinatorial fusions could modulate the gene drive activity within a cell population [110].…”

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][25][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][22][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][25][26].…”

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 ].…”