Brent Carroll, BS

Alternative splicing increases the diversity of gene expression by regulating exon-skipping and inclusion events to produce different mRNAs from the same DNA gene locus. These different mRNA sequences can be translated into proteins called isoforms, which can have different functions. The majority of alternative isoforms remain functionally uncharacterized due to the technical limitations of perturbing specific isoforms in high-throughput. One high-throughput technique to determine gene function uses DNA-targeting CRISPR-Cas9 (Cas9). By linking guide RNA sequences to phenotypic changes, such as cell survival and growth, gene functions can be inferred in high-throughput. However, Cas9 is fundamentally unable to target individual isoforms of a given gene. In response to this limitation, we developed a high-throughput screening platform using RNA-targeting CRISPR-Cas13 (Cas13) to perturb individual isoforms in high-throughput. We tested a variety of variables to optimize isoform-specific knockdown and found that variables including Cas13 variants, guide RNA sequences, and nuclear export/localization signal tags each affect knockdown efficiency. With these optimizations, researchers will be able to perform high-throughput screens on isoform-specific targets. Characterizing and optimizing RNA-targeting CRISPR-Cas13 systems in an isoform-aware manner lays the foundation for genomics researchers to begin to decode the “dark matter” of alternative isoforms.