A Multiple Gene Drive System

Ferdinand Nanfack Minkeu is a graduate student at the Pasteur Institute in Paris, France where he studies host-pathogen interactions in African malaria mosquitos and is also interested in transgenic tools and technologies for vector control.

Yan & Finnigan, (2018) recently published a paper in Scientific Reports describing an artificial multi-locus gene drive system by using a single Cas9 and three guide RNA (gRNA) in the budding yeast Saccharomyces cerevisiae. Nuclease-based gene drives do not follow the typical Mendel patterns of inheritance and lead quickly to the spread of gene drive elements through a given population.

Gene drive technology may be used to control vector diseases, crop pests and other harmful species. For instance, population suppression or replacement with anti-Plasmodium genes into malaria vectors (Gantz et al., 2015; Eckhoff et al., 2017). The application of this approach requires a good design and the questions about biosafety, ethic, containment, control and others should be addressed. Budding yeast is a powerful model system that can permit testing and optimizing of innovative gene drive designs, control mechanisms, and reversal strategies prior to their introduction into species that are more difficult to manipulate.

Saccharomyces cerevisiae

Yan & Finnigan’s (2018) method is based on what they call a minimal gene drive (MGD) which can be used to carry out the precise replacement of multiple native genes. (Fig.1)

Their strategy involves the use of three gRNAs and a single Cas9 locus and leads to the cleavage of the HIS3, SHS1 and DNL4 loci. To safeguard against these gene drive systems escaping the laboratory the authors constructed a line in which each of the target loci has been replaced with sequences that are unique to the transgenic line and not found in yeasts in nature.  Each target locus upon integration of the minimal gene drive element will result in a unique selectable/screenable phenotype. They also created a three-locus strain with the three drive elements.  Drive #1 contained an induceable Cas9 locus and a guide RNA-expressing transgene.  Drives #2 and #3 consisted of only of unique guide RNA expressing transgenes.  Mating between the triple MGD strain and the triple target strain produced diploids in which Cas9 was galactose-dependent.

Multilocus gene drive with a single source of Cas9. In Yan and Finnegan (2018) each target has been engineered to contain non-yeast sequences that were targets of the guide RNAs associate with each drive as a biosafety precaution to avoid unintentional introduction of gene drives into yeasts outside the laboratory.
Image Credit: Ferdinand Nanfack.

Their results showed that in the absence of nuclease expression, 95% of yeast colonies showed no evidence of movement of the gene drive, but five hours after incubation in galactose, resistant colonies become sensitive, meaning that the target genes were being mutagenized by the integration of the drive elements into their target sites.

While the frequency of drive was high this method is not yet perfect because 5% of the resulting yeast colonies are still resistant, indicating that the gene drive elements did not invade some target loci. This latter can be explained by poor or lost expression of gRNA, nonhomologous end joining repair and others.

Unlike single locus drive systems, three locus systems can be more efficient and they allow accurate introduction of additional endogenous genes or genetically modified alleles as well as being better able to avoid the evolution of resistance. Moreover, the use of a single source of Cas9 to drive movement of the minimal gene drives facilitates removal or inactivation of Cas9 by using an anti-drive measure in cases where terminating the spread of a gene drive is warranted.

Duina AA, Miller ME, Keeney JB. (2014). Budding Yeast for Budding Geneticists: A Primer on the Saccharomyces cerevisiae Model System. Genetics, 197(1): 33–48.

Gantz VM, Jasinskiene N, Tatarenkova O, Fazekas A, Macias VM, Bier E, James AA. (2015). Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. PNAS, 112(49):E6736-43.

Philip A. Eckhoff, Edward A. Wenger, H. Charles J. Godfray, and Austin Burt. (2017). Impact of mosquito gene drive on malaria elimination in a computational model with explicit spatial and temporal dynamics. PNAS, 114 (2) E255-E264

Yan Y & Finnigan GC. (2018). Development of a multi-locus CRISPR gene drive system in budding yeast. Scientific reports, 8 (1) 17277.





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