High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects

Abstract

CRISPR-Cas13 systems have recently been employed for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has imposed a major barrier for their in vivo applications. We designed a dual-fluorescent reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants (including Cas13d and Cas13X) exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. Importantly, high-fidelity Cas13 variants show comparable RNA knockdown activity with wild-type Cas13 but no detectable collateral damage in transgenic mice and adeno-associated virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effect are now available for targeted degradation of RNAs in basic research and therapeutic applications.

Polyvalent guide RNAs for CRISPR antivirals

“…Although the prospect of interactions with the host genome or transcriptome is bioinformatically minimized with our pgRNA design algorithm, potential off-target effects will need to be experimentally evaluated very carefully for each potential viral target and (p)gRNA sequence to ensure their safety. Another potential concern has been raised with regards to potential toxicity that can occur as a result of Cas13’s collateral activity ( Özcan et al., 2021 ; Tong et al., 2022 ), although in our study plants appeared phenotypically healthy. This toxicity has been observed in animal cell lines and transgenic animals under conditions where both Cas13 and its target are highly expressed during for targeted RNA knock-down by Cas13 for long periods of time, conditions not likely to be necessary for antiviral applications.…”

“…After three days, plants expressing one of six different monovalent gRNAs showed viral RNA levels in their leaves reduced in general to approximately 5–25% of plants expressing Cas13 and either a non-targeting gRNA (gRNA-NT) or no gRNA ( Figures 2 C–2E). While concerns have been raised regarding the potential toxicity of long-term expression of Cas13d as a result of “collateral activity” (see below) ( Tong et al., 2022 ), Cas13d-treated plants appeared phenotypically healthy over the course of the experiments ( Figure 2 C). Plants expressing a single monovalent gRNAs exhibited in general less viral replication suppression than those expressing a single pgRNA, which were able to robustly suppress viral spread ( Figure 2 C) and viral gene expression by >97% relative to GFP mRNA levels in plants expressing a gRNA-NT ( Figures 2 D, 2E, S4 , and Table S7 : RT-qPCR of GFP mRNA in N. bethamiana after transient expression of TRBO-GFP; RfxCas13d; and gRNAs, pgRNAs, or non-targeting (NT) gRNAs).…”

“…This toxicity has been observed in animal cell lines and transgenic animals under conditions where both Cas13 and its target are highly expressed during for targeted RNA knock-down by Cas13 for long periods of time, conditions not likely to be necessary for antiviral applications. It remains to be seen whether or not Cas13’s collateral activity is necessary for its antiviral effects in vivo but, if not, engineered Cas13 variants with targeted activity but minimized collateral activity ( Tong et al., 2022 ) or alternative RNA-targeting CRISPR effector Cas7-11 that does not exhibit collateral activity ( Özcan et al., 2021 ) could provide potentially safer opportunities. In addition, although previous studies in human cell lines did not find evidence of elevated mutation rates in CRISPR-targeted viruses ( Freije et al., 2019 ), our laboratory is currently evaluating “long-term” effects of CRISPR antivirals on the mutation rate of viral targets, as this will be another critical consideration; we suspect that use of pgRNAs can further help suppress the potential for viral mutagenic escape.…”

Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

“…On the other hand, collateral degradation of bystander RNAs by CRISPR-Cas13 system has limited its applications in vivo. Hence high-fidelity Cas13 variants with minimal collateral effects have been developed, which could further elevate their utility for targeted degradation of RNAs in basic research and therapeutic applications 223 .…”

Applications of CRISPR/Cas13-Based RNA Editing in Plants

“…One major limitation is the collateral activity of Cas13 that causes non-specific RNA cleavage [ 76 , 77 , 78 ]. To overcome this limitation, Tong et al [ 79 ] recently developed high-fidelity Cas13 variants with a dual-fluorescent-reporter system to detect and screen Cas13 variants in mammalian cells, providing efficient RNA targeting and minimizing collateral effects. However, no such collateral activity has been observed in plants yet.…”