UC Berkeley

RNA mediates a myriad of functions, from encoding genetic information to catalyzing enzymatic reactions required for life. The discovery of RNA-guided, RNA-targeting CRISPR effectors allows researchers to target RNA molecules in a sequence specific manner, and this work details two applications. First, I describe the use of Leptotrichia buccalis Cas13a (LbuCas13a) in conjunction with an isothermal amplification protocol for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SaRS-CoV-2) RNA from contrived and clinical samples following an extraction-free sample lysis. This diagnostic protocol, named DISCoVER (DIagnostics with Coronavirus Enzymatic Reporting), detects positive samples with 94% sensitivity and 100% specificity, as validated by qPCR. Next, I describe the use of catalytically inactive Cas13d from Ruminococcus flavefaciens XPD3002 (dCasRx) and Cas7-11 from Desulfonema ishimotonii to target pre-mRNA molecules to facilitate kilobase scale edits, termed RESPLICE (RNA-guided trans-splicing with Cas editors). Pre-mRNA molecules in eukaryotic cells undergo splicing to stitch exons together, allowing for a diverse and dynamic set of protein products that execute a gene’s biological functions. Inspired by the natural process of trans-splicing that joins exons from two distinct primary transcripts in trypanosomes, RESPLICE co-localizes the trans-splicing module (TSM) to the target and inhibits the cis-splicing reaction to allow the spliceosome to incorporate the cargo from the TSM into the target. I demonstrate efficient, specific and programmable trans-splicing of multi-kilobase cargoes, achieving up to 45% trans-splicing efficiency in bulk and 90% efficiency when sorting for high effector expression. This work demonstrates uses of RNA-guided, RNA targeting CRISPR effectors towards diagnostics and transcriptome engineering.