Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens

Abstract

Summary: The recent characterization of RNA-targeting CRISPR nucleases has enabled diverse transcriptome engineering and screening applications that depend crucially on prediction and selection of optimized CRISPR guide RNAs (gRNAs). Previously, we developed a computational model to predict RfxCas13d gRNA activity for all human protein-coding genes. Here, we extend this framework to six model organisms (human, mouse, zebrafish, fly, nematode, and flowering plants) for protein-coding genes and noncoding RNAs (ncRNAs) and also to four RNA virus families (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2], HIV-1, H1N1 influenza, and Middle East respiratory syndrome [MERS]). We include experimental validation of predictions by testing knockdown of multiple ncRNAs (MALAT1, HOTAIRM1, Gas5, and Pvt1) in human and mouse cells. We developed a freely available web-based platform (cas13design) with pre-scored gRNAs for transcriptome-wide targeting in several organisms and an interactive design tool to predict optimal gRNAs for custom RNA targets entered by the user. This resource will facilitate CRISPR-Cas13 RNA targeting in model organisms, emerging viral threats to human health.

Keywords

• Cas13
• CRISPR
• model organisms
• RNA viruses
• on-target efficiency prediction