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Review
. 2019 Jun;35(6):446-456.
doi: 10.1016/j.tig.2019.03.009. Epub 2019 Apr 27.

Three New Cs for CRISPR: Collateral, Communicate, Cooperate

Andrew Varble  1 , Luciano A Marraffini  2
Affiliations
Review

Three New Cs for CRISPR: Collateral, Communicate, Cooperate

Andrew Varble et al. Trends Genet. 2019 Jun.

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated (cas) genes provide protection against invading phages and plasmids in prokaryotes. Typically, short sequences are captured from the genome of the invader, integrated into the CRISPR locus, and transcribed into short RNAs that direct RNA-guided Cas nucleases to the nucleic acids of the invader for their degradation. Recent work in the field has revealed unexpected features of the CRISPR-Cas mechanism: (i) collateral, nonspecific, cleavage of host nucleic acids; (ii) secondary messengers that amplify the immune response; and (iii) immunosuppression of CRISPR targeting by phage-encoded inhibitors. Here, we review these new and exciting findings.

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Conflict of interest statement

Declaration of interests. L.A.M. is a cofounder and Scientific Advisory Board member of Intellia Therapeutics, and a co-founder of Eligo Biosciences.

Figures

Figure 1.
Figure 1.. Collateral nucleic acid degradation by Cas nucleases and CRISPR secondary messenger signaling.
(A) RNA targeting by the type VI effector Cas13a. Cas13a possesses two HEPN domains (green circle, H). Binding of target RNA (red line) to the crRNA (black line) induces a conformational shift that brings the domains in close proximity to form an active HEPN site (green star). The active site then cleaves RNA in a sequence-independent manner. (B) Nucleic acid targeting by the prototypical type V effector protein, Cas12a. The RuvC domain (green circle, R) is responsible for the nucleolytic activity of Cas12a. Before target recognition, this domain is obscured by the Rec lobe. Upon binding of either double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) targets (blue lines) to the crRNA (black line), the effector undergoes a conformational shift that exposes and activates the RuvC domain (green star) to license cleavage of the target DNA and non-specific cleavage of ssDNA. (C) Nucleic acid targeting and secondary messenger signaling by the type III-A CRISPR-Cas system. Upon recognition of a target transcript (red line) to the crRNA (black line), the type III complex activates multiple effector functions. The HD domain of the Cas10 subunit (green circle, H) is able to non-specifically cleave ssDNA (blue line) from the transcription bubble. The Palm domain of Cas10 (green circle, P) is also activated and converts ATP into 4 or 6 member rings of cyclic oligoadenylates (cOA). cOA then acts as a secondary messenger and activates Csm6 by binding to its CARF domain causing activation of the HEPN domain (green circle, inactive, or green star, active; H), unleashing non-specific RNA cleavage. Additionally, not depicted, a subunit of the type III complex, Csm3, is a specific RNase that cleaves the bound transcript.
Figure. 2.
Figure. 2.. Mechanisms of phage CRISPR inhibitors.
(A) CRISPR inhibitors of type I-F systems. The first class of inhibitors bind to the type I-F Cascade complex and prevent the binding of dsDNA. AcrIF1 binds the between Cas7f subunits at regular intervals, locking the complex in a formation that cannot bind dsDNA. AcrIF2 binds between the Cas7f and Cas8f subunits and AcrIF10 binds between Cas8f and Cas5f. Both inhibitors prevent the entry of DNA into the targeting complex. After the crRNA within Cascade (black line) has bound to its DNA target (blue line), the complex recruits Cas3, a DNase that degrades the target DNA. AcrIF3 forms a dimer that prevents recruitment of Cas3 to the targeting complex and blocks DNA entry into the Cas3 nucleolytic site. (B) CRISPR inhibitors of type II systems. AcrIIA2 and AcrIIA4 are inhibitors of the type II-A Cas9 nuclease. During target search Cas9 scans DNA for protospacer adjacent motif (PAM), which recognition is the first step in licensing DNA cleavage. AcrIIA2 and AcrIIA4 both bind the portion of Cas9 that recognizes the PAM, thus blocking DNA loading into the nuclease. AcrIIC2 binds the bridge helix of Cas9 to interfere with crRNA/tracrRNA binding. AcrIIC3 is a type II-C Cas9 inhibitor that causes Cas9 dimerization and also prevents DNA loading. AcrIIC1 binds the HNH domain, preventing cleavage of the target strand of DNA, along with blocking the conformational shift required to license RuvC-mediated cleavage of the non-target strand. (C) Phage cooperation is required for effective CRISPR inhibition by Acrs. Of the currently identified Acrs, none are injected during phage infection, indicating they must be transcribed and translated post-injection. Therefore, the host is immuno-suppressed with every infection wave. The success of inhibition depends on three factors: the multiplicity of infection (MOI), the strength of the Acr and the strength of CRISPR targeting.
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