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

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.

Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

“…Complete activation of Cas13 by perfect crRNA:target pairing often induces a collateral effect that leads to nonspecific cleavage of bystander ssRNA. Such collateral effect was originally observed in bacterial cells for Cas13a (Abudayyeh et al ., 2016), but was recently reported for more Cas13 effectors including Cas13d (Ai et al, 2022; Kelley et al, 2021; Li et al, 2022; Shi et al, 2021; Tong et al, 2021; Wang et al, 2019b). Compared with collateral cleavage, intrinsic RNA targeting we described here is fundamentally distinct in the following ways: (1) intrinsic RNA targeting is more prevalent as long as Cas13 protein is present, whereas collateral effect requires the presence and abundance of fully activated Cas13:crRNA:on-target RNA ternary complex; (2) more RNase domains and activities of Cas13 may be involved in intrinsic RNA targeting, while collateral RNA cleavage is exclusively mediated by the two HEPN domains of Cas13; (3) the cleavage effect of intrinsic RNA targeting is comparatively subtle and selective; whereas collateral RNA cleavage is drastic and nonspecific; (4) the influence of intrinsic RNA targeting is not limited to RNA cutting but may extend to broader RNA metabolism processes such as RNA translation or localization control, whereas collateral effect is only restricted to RNA cleavage; and (5) the phenotypic effect of intrinsic RNA targeting can be latent (e.g., lentiviral defect) or unspecified depending on given transcriptome milieu and target cell types, whereas collateral effect often causes apparent cellular toxicity (Figure S6H).…”

Precise Transcript Targeting by CRISPR-Csm Complexes

“…Other RNA-targeting CRISPR-Cas systems such as Cas13 suffer from severe cytotoxic effects due to inherent trans -cleavage activity of the Cas effector (Q. Wang et al 2019; Özcan et al 2021; Ai, Liang, and Wilusz 2022; Tong et al 2021; Shi et al 2021). Type III systems do not exhibit such trans -activity and are thus poised to offer robust RNA KD without such toxicity.…”

“…More importantly, RNA KD was not accompanied by detectable cytotoxicity, unlike Cas13-based KD which suffers from inherent trans-cleavage activity (Q. Wang et al 2019;Özcan et al 2021;Ai, Liang, and Wilusz 2022;Tong et al 2021).…”

“…While simpler introduce into eukaryotes, Cas7-11’s demonstrated RNA KD efficiency was 50-75% for most targets, making it less practical as a tool. Meanwhile, a recent pre-print of engineered high-fidelity (hf)Cas13 variants reports to have mitigated trans- cleavage activity (and thus cytotoxicity) while preserving on-target activity (Tong et al 2021)--though a mechanistic explanation for this remains unclear. Both Cas7-11 and hfCas13 await further characterization before widespread use.…”

“…Only recently, however, has evidence mounted that Cas13 exhibits trans -cleavage activity in eukaryotic cells, causing cytotoxicity and/or cell death (Q. Wang et al 2019; Özcan et al 2021; Ai, Liang, and Wilusz 2022; Tong et al 2021; Shi et al 2021). The convolution of cis- and trans-RNA cutting effects have made it difficult to interpret results obtained using Cas13, and call into question its utility as a tool for specific RNA knockdown.…”