Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.

Integration of prophages into CRISPR loci remodels viral immunity inStreptococcus pyogenes

“…2F). This differs from the mode of action of previously described anti-CRISPRs that broadly turn off CRISPR function (Davidson et al, 2020), and can provide a selective advantage for ΦAP1.1 (and related phages), which could exploit integration to neutralize spacers that target its genome, but at the same time maintain the immunity provided by other spacers against competitor phages, promoting host survival. Additionally, these lysogens could also retain the ability to acquire new spacers in the first position of the CRISPR array against new invaders.…”

“…Cas immunity is most commonly evaded by phages containing target mutations that prevent recognition and cleavage by Cas9 (Deveau et al, 2008;Gasiunas et al, 2012;Jinek et al, 2012). In addition, some phages also produce anti-CRISPR (Acr) proteins that specifically inhibit Cas nucleases (Davidson et al, 2020). Although none have been isolated from S. pyogenes phages, multiple Acrs that prevent the function of different type II-A Cas9 nucleases with diverse mechanisms of inhibition have been reported (Eitzinger et al, 2020;Forsberg et al, 2019;Hynes et al, 2017;Mahendra et al, 2020;Rauch et al, 2017;Uribe et al, 2019).…”

Discovery of multiple anti-CRISPRs uncovers anti-defense gene clustering in mobile genetic elements

“…Former mechanistic characterizations of Acr proteins have revealed a remarkable diversity of inhibitory functions (Davidson et al, 2020). Interestingly, relatively low MWs and pIs have been reported for Acrs inhibiting CRISPR-Cas systems via DNA mimicry (Jiang et al, 2019;Liu et al, 2019), suggesting a putative link between low MW/pI values and mechanistic inhibitory function.…”

“…types II, III and V) (Pawluk, Amrani, et al, 2016;Rauch et al, 2017;Marino et al, 2018;Watters et al, 2018;Bhoobalan-Chitty et al, 2019;Athukoralage et al, 2020), including some on non-phage MGEs . Previous structural and biochemical characterization of Acr proteins has revealed a diverse range of inhibitory activities, including interference with crRNA loading, inhibition of target DNA recognition, and inhibition of DNA cleavage, among others (Davidson et al, 2020). Apart from sharing a typically low molecular weight, Acrs lack conserved sequence and structural features, thus rendering de novo prediction largely impractical with current methods.…”

Types I and V Anti-CRISPR Proteins: From Phage Defense to Eukaryotic Synthetic Gene Circuits

“…Type I and type V-A anti-CRISPR proteins carry out their action mainly by preventing Cascade/Cas12a-crRNA complex from recognizing and binding the target DNA or by abolishing DNA cleavage after inactivating the Cas effector. However, enzymatic behaviors have been reported too (Davidson et al, 2020;Wiegand et al, 2020).…”