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

In brief To accelerate CRISPR-based targeting of RNA, Guo et al. present a resource with optimized RfxCas13d guide RNAs (gRNAs) to target messenger RNAs and noncoding RNAs in six common model organisms and four RNA virus families. An accompanying open access web-based platform and design tool enable optimal gRNA design for any RNA target.

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

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.

The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types.

A novel variant of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) has recently emerged and rapidly surpassed others in prevalence. This mutation is in linkage disequilibrium with an ORF1b protein variant (P314L), making it difficult to discern the functional significance of the Spike D614G mutation from population genetics alone. Here, we perform site-directed mutagenesis on wild-type human-codon-optimized Spike to introduce the D614G variant. Using multiple human cell lines, including human lung epithelial cells, we found that the lentiviral particles pseudotyped with Spike D614G are more effective at transducing cells than ones pseudotyped with wild-type Spike. The increased transduction with Spike D614G ranged from 1.3- to 2.4-fold in Caco-2 and Calu-3 cells expressing endogenous ACE2 and from 1.5- to 7.7-fold in A549ACE2 and Huh7.5ACE2 overexpressing ACE2. Furthermore, trans-complementation of SARS-CoV-2 virus with Spike D614G showed an increased infectivity in human cells. Although there is minimal difference in ACE2 receptor binding between the D614 and G614 Spike variants, the G614 variant is more resistant to proteolytic cleavage, suggesting a possible mechanism for the increased transduction.