ABSTRACT Prokaryotic CRISPR-Cas systems are highly vulnerable to phage-encoded anti-CRISPR (Acr) factors. How CRISPR-Cas systems protect themselves remains unclear. Here, we uncovered a broad-spectrum anti-anti-CRISPR strategy involving a phage-derived toxic protein. Transcription of this toxin is normally reppressed by the CRISPR-Cas effector, but is activated to halt cell division when the effector is inhibited by any anti-CRISPR proteins or RNAs. We showed that this abortive infection-like effect efficiently expels Acr elements from bacterial population. Furthermore, we exploited this anti-anti-CRISPR mechanism to develop a screening method for specific Acr candidates for a CRISPR-Cas system, and successfully identified two distinct Acr proteins that enhance the binding of CRISPR effector to non-target DNA. Our data highlight the broad-spectrum role of CRISPR-repressed toxins in counteracting various types of Acr factors, which illuminates that the regulatory function of CRISPR-Cas confers host cells herd immunity against Acr-encoding genetic invaders, no matter they are CRISPR-targeted or not.

CRISPR-repressed toxin-antitoxin provides population-level immunity against diverse anti-CRISPR elements