A comprehensive analysis and resource to use CRISPR-Cas13 for broad-spectrum targeting of RNA viruses

Abstract

Highlights d A web resource for using CRISPR-Cas13 to target broad RNA virus families d A bioinformatic tool to analyze efficiency and specificity of virus-targeting crRNAs d A minipool of crRNAs can target 90% of human-infectious RNA viruses d Validated crRNAs are predicted to target newly emerging SARS-CoV-2 variants

Genome Editing Technologies as Cellular Defense Against Viral Pathogens

“…Specifically, they observed that the decrease in the percentage of SARS-Cov-2 reporter-positive cells correlated significantly with the predicted targeting efficiency score of crRNAs and with the viral RNA abundance. These results validated their prediction algorithm for the targeting efficiency of crRNAs for the viral genome (Lin et al, 2021).…”

“…The in silico analysis showed that a minimal set of 14 crRNAs can target >90% of human-tropic viral genomes. Furthermore, by co-transfecting plasmids containing one of five predicted crRNAs and SARS-CoV-2 reporters into A549 lung epithelial cells, Lin et al (2021) validated the PAC-MAN strategy. Specifically, they observed that the decrease in the percentage of SARS-Cov-2 reporter-positive cells correlated significantly with the predicted targeting efficiency score of crRNAs and with the viral RNA abundance.…”

“…Additionally, they utilized bioinformatics analysis to predict groups of CRISPRassociated RNAs to target all sequenced coronaviruses, though the effectiveness and in vivo safety of this strategy need to be examined before putting it into clinical use (Abbott et al, 2020). To expand their work, Lin et al (2021) applied the PAC-MAN strategy to a broad spectrum of human-or livestock-tropic RNA viruses. The in silico analysis showed that a minimal set of 14 crRNAs can target >90% of human-tropic viral genomes.…”

“…While individual crRNA showed an ability to target almost 99% of SARS-CoV-2 isolates, the pooling of the five experimentally validated crRNAs could target all of the SARS-CoV-2 variants as identified in GISAID on February 3, 2021 (Lin et al, 2021). Moreover, Lin et al (2021) have rolled out an online resource 1 for the use of CRISPR/Cas13 to target RNA viruses, which when combined with rapid field-deployable virus sequencing (Bi et al, 2021) could greatly speed up the design of genome editing tools for combating emerging viruses. These proof-of-principle studies highlight the versatility and flexibility of CRISPR as an intracellular defense against many types of RNA viruses, including but not limited to SARS-CoV-2.…”

Designing viral diagnostics with model-based optimization

“…These models predict on-target cleavage by measuring knockdown efficacy or indel frequency, and several 18,19 have focused on predictions for CRISPR-Cas13 enzymes using manually-defined features; Cas13 enzymes have applications in diagnostics and are our modeling focus in this work. Subsequent studies 20,21 have applied one of these models 18 to improve Cas13d guide design for viral RNA knockdown, having use in antivirals. To our knowledge, our modeling is the first that applies deep learning to predict Cas13 guide activity.…”