CRISPR-repressed toxin–antitoxin provides herd immunity against anti-CRISPR elements

Prokaryotic clustered regularly interspaced short palindromic repeat (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 repressed 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 nontarget DNA. Our data highlight the broad-spectrum role of CRISPR-repressed toxins in counteracting various types of Acr factors. We propose that the regulatory function of CRISPR–Cas confers host cells herd immunity against Acr-encoding genetic invaders whether they are CRISPR targeted or not.