A target expression threshold dictates invader defense and autoimmunity by CRISPR-Cas13

Abstract

SUMMARYImmune systems must recognize and clear foreign invaders without eliciting autoimmunity. CRISPR-Cas immune systems in prokaryotes manage this task by following two criteria: extensive guide:target complementarity and a defined target-flanking motif. Here we report an additional requirement for RNA-targeting CRISPR-Cas13 systems: expression of the target transcript exceeding a threshold. This finding is based on targeting endogenous non-essential transcripts, which rarely elicited dormancy through collateral RNA degradation. Instead, eliciting dormancy required over-expressing targeted transcripts above a threshold. A genome-wide screen confirmed target expression levels as the principal determinant of cytotoxic autoimmunity and revealed that the threshold shifts with the guide:target pair. This expression threshold ensured defense against a lytic bacteriophage yet allowed tolerance of a targeted beneficial gene expressed from an invading plasmid. These findings establish target expression levels as a third criterion for immune activation by RNA-targeting CRISPR-Cas systems, buffering against autoimmunity and distinguishing pathogenic and benign invaders.HIGHLIGHTSCas13-induced dormancy requires RNA target levels to exceed an expression thresholdThe expression threshold can prevent cytotoxic self-targeting for endogenous transcriptsThe threshold shifts depending on the CRISPR RNA guide:target pairThe threshold allows cells to distinguish pathogenic and benign infections

The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model

“…Similarly, Yang group also found that LwaCas13a, RfxCas13d, and Cas13X.1 exhibited weak collateral activity when targeting transiently overexpressing mCherry, but not endogenous genes, using EGFP stably expressed in HEK293T as the indicator of collateral effects [ 14 ], which gives us a hint that the collateral activity of Cas13 may relate with the abundance of target RNA. Besides, in bacteria, Cas13-induced dormancy requires target RNA levels to exceed an expression threshold [ 40 ]. And in vitro experiments proved that collateral activity of Cas13 is positively correlated with the abundance of target RNA [ 6 , 9 ].…”

Improved prediction of bacterial CRISPRi guide efficiency from depletion screens through mixed-effect modeling and data integration

“…We have shown that this model improves on the previous state-of-the-art using both gene-wise cross-validation on our training data as well as a fully independent screen of guides targeting purine biosynthesis genes essential in minimal media. These investigations provide a blueprint for developing similar predictive models, both for other CRISPR-Cas systems (Vialetto et al, 2021) and technologies, as well as for CRISPRi in different bacteria where design rules may vary. We have made a web server for predicting CRISPRi guide efficiency using our MERF publicly available at: https://ciao.helmholtz-hiri.de.…”

“…While we focused here on applications of CRISPRi with dCas9 in E. coli , the techniques we have developed are in principle generic and could be extended to CRISPRi with any catalytically-dead nuclease in any bacterium of interest, or even to entirely different CRISPR systems. For instance, we recently applied the same basic methodology to investigate the features underlying autoimmune activation of Cas13 targeting cellular RNA (Vialetto et al, 2021). It is becoming increasingly clear that the performance of CRISPRi depends on both genetic background and the specific Cas protein used.…”