The lone virus dies but the pack survives

The bacteria use CRISPR- Cas system as immune defense in response of viral infection. CRISPR-Cas immunity generally occurs through three steps: adaptation, expression, and interference. During the first one, fragments of invading foreign DNA – protospacers – are integrated into the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) array. While expression, the CRISPR array is transcribed and processed to yield single repeat-spacer RNA known as CRISPR- RNA (crRNA). crRNAs are subsequently complexed with one or more Cas (CRISPR- associated) proteins to form ribonucleoprotein surveillance complexes. During interference, these complexes search foreign nucleic acid through complementarity with the crRNAs and cut it.

Before the discovery of anti-CRISPR proteins, the acquisition of point mutations within the protospacer or the PAM (protospacer adjacent motif) or seed sequence, a region of the protospacer that must have perfect complementarity to the crRNA, have been shown to be enough for phages to escape CRISPR-Cas immunity. Since CRISPR-Cas system is adaptive and can re-acquire new spacers, it seemed that bacteria have won. In 2013, the first active counter-mechanism to CRISPR-Cas immunity, anti-CRISPR proteins, was identified within a small group of closely relate Pseudomonas aeruginosa phages. To date, have been classified a total of 40 distinct families of anti-CRISPR proteins. The peculiarity of Acr proteins is they share no common sequence or motif among them. The great diversity is both structural and functional. Anti-CRISPRs may be able to prevent Cas proteins from acquiring new spacers. They could potentially also block transcription of the CRISPR array or processing of the transcript into mature crRNAs. The anti-CRISPR proteins that inhibit the interference bind directly to the effector protein complex and could thereby inhibit one of three steps: crRNA loading, target DNA binding, or target DNA cleavage. Anti-CRISPRs that obstruct crRNA loading have not yet been reported, but anti-CRISPRs that block both target DNA binding and cleavage have [1].

The discovery of type V-A anti-CRISPR proteins was an exciting advance, as Cas12a, the effector protein in type V-A CRISPR-Cas systems, is an attractive alternative to Cas9 for genome editing purposes. A new study recently published in Nature Struct. Mol. Biol by Knott and collaborators [2] shows that AcrVA1 uses enzymatic activities previously unobserved to inactivate CRISPR-Cas12a system. The formation of the Cas12a-crRNA complex is essential for targeting and cutting viral DNA. The nuclease Cas12a catalyzes both single-turnover target DNA cutting (cis-cleavage) and multiple-turnover non-target ssDNA cutting (trans-cleavage). After the cis-cleavage, the PAM-proximal DNA remains bound to the Cas12a-crRNA complex: this keep Cas12a active which permits unusual trans cleavage. Knott and collaborators wondered in the first instance whether AcrVA1, AcrVA4, AcrVA5 could prevent the formation of the complex, then what their effect was once the complex was already formed (strategy that may have evolved to disable an activated and trans-cleaving Cas12a). The results showed that AcrVA1, although it is not a RNAse, cuts the guide crRNA; the cleaved guide fragment and AcrVA1 are released, the inhibitor then can turn over and cleave crRNA in other copies of the Cas12a effector complex (Fig. 1a). The shortened crRNA still bound to Cas12a is not competent for binding target DNA, as it lacks complementarity to the target region of the dsDNA. In addition to that, the limited length of the crRNA could prevent the formation of a hybrid structure stable.  AcrVA1 triggers endoribonucleolytic truncation of both mature and pre-crRNA in the presence of three Cas12a orthologs (Moraxella bovoculi, Acidaminococcus sp., Lachnospiracea bacterium); it alone lacks RNA-cleavage activity, but it robustly cleaves the crRNA within the effector complex. At sub-stoichiometric concentrations of AcrVA1, crRNA cleavage is still observed at maximal levels. AcrVA1 activity is multiple turnover where cleavage of a crRNA will inactivate Cas12a–crRNA complexes [3]. Neither residues in the Cas12a active site nor metal ions (MgCl₂ or EDTA) are required for the AcrVA1-dependent truncation of crRNA, although the exact mechanism of AcrVA1’s activity remains unclear. At present, is know that the activity of AcrVA1 is independent of spacer’s sequence or length. The authors did not perform experiments to figure out whether AcrVA1 recognizes a specific site of crRNA. Notably, AcrVA1 has previously been shown to potently inhibit a broad range of Cas12a orthologs suggesting that this activation can occur across several Cas effector sequences [4]. The prowess of AcrVA1 to inhibit all three Cas12a orthologs used, proposes that it might exploit an evolutionary conserved domain of Cas12a. In fact, the PAM-interacting domain is severely required for process of inhibition.