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Evaluating Factors Affecting the Performance of the gRNA Component of a Gene Drive

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Abstract

CRISPR-based gene drives are promising technologies that breach Mendelian inheritance rules and allow for the rapid propagation of desired traits into target wild populations. These tools offer novel potential applications ranging from vector-borne disease control to island conservation. However, current gene drives do not always perform efficiently, which begs for the improvement of this technology in order to make gene drives a feasible method to engineer wild populations. Here, I first evaluate the effects of gRNA variables that could affect CRISPR-Cas9 gene drive performance: GC content and protospacer length. In this part of my thesis, I tested six different gRNAs with varying GC content (50%, 80%) and length (17nt, 20nt, 22nt) in Drosophila and determined that the 20nt-protospacer gRNAs have the highest gene drive efficiency, followed by 17nt and 22nt lengths, where the longer gRNA seems to impair the gene drive efficiency more severely at the two locations tested.

In addition, we evaluated the relationship between gRNA efficiency and a maternal effect affecting this technology--where gene drive inheritance drastically decreases when the Cas9 and gRNA are co-inherited from the mother. Cage experiments over 12 generations show that less efficient gRNAs are able to propagate the gene drive component at higher rates throughout the populations, as compared to its higher-efficiency counterpart. Future CRISPR-based gene drive research should consider the findings described here, as the use of less efficient gRNAs could alleviate the effects of maternal inheritance, and different gRNA lengths could tune drive efficiency in future applications.

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This item is under embargo until September 11, 2023.