Broad-spectrum enzymatic inhibition of CRISPR-Cas12a

Knott, Gavin J.; Thornton, Brittney W.; Lobba, Marco; Liu, Jun Jie; Al-Shayeb, Basem; Watters, Kyle E.; Doudna, Jennifer A.

doi:10.1038/s41594-019-0208-z

Cited by 104 publications

(118 citation statements)

References 42 publications

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“…All are activated by cA4 and would thus be deactivated by AcrIII-1. Recently, two other Acr proteins with enzymatic functions have been described: AcrVA1 which catalyses crRNA cleavage of Cas12a 35 and AcrVA5, which acetylates the PAM-sensing site of Cas12a 36 . Both, however, target a protein (or protein:nucleic acid complex), implying a requirement for specific interactions that could be evaded by sequence variation.…”

Section: Figure 2 Acriii-1 Rapidly Degrades Ca4 To Linear Products (A)mentioning

confidence: 99%

A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

Athukoralage

McMahon

Zhang

et al. 2019

Preprint

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The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger 1-3 , activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction 4,5 . Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions 6,7 . Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise the type III CRISPR defence system. The AcrIII-1 family has a broad host range as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.Type III CRISPR-Cas systems synthesise the signalling molecule cyclic oligoadenylate (cOA) from ATP 1,2 when they detect viral RNA. cOA molecules are synthesised with a range of ring sizes with 3-6 AMP subunits (denoted cA3, cA4 etc.) by the cyclase domain of the Cas10 protein [1][2][3]9,10 . cOA binds to a specific protein domain, known as a CARF (CRISPR Associated Rossman Fold) domain. CARF domains are found fused to a variety of effector domains that are known or predicted to cleave RNA, DNA, or function as transcription factors 11 . The best characterised CARF protein family is the Csx1/Csm6 family of HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) ribonucleases, which are activated by cOA binding and cleave RNA with minimal sequence dependence 1-3 . A number of studies have demonstrated that the cOA signalling component of type III systems is crucial for effective immunity against viruses 4,12-15 , highlighting the importance of this facet of CRISPR immunity.Recently, we identified a cellular enzyme in Sulfolobus solfataricus, hereafter referred to as the Crn1 family (for "CRISPR associated ring nuclease 1"), that degrades cA4 molecules and thus deactivates the Csx1 ribonuclease in vitro 16 . These enzymes exhibit very slow kinetics, and are thought to act by mopping up cA4 molecules in the cell without compromising the immunity provided by the type III CRISPR system. Unsurprisingly, viruses have responded to the threat of the CRISPR system by evolving a range of anti-CRISPR (Acr) proteins, which are used to inhibit and overcome the cell's CRISPR defences (reviewed in 17 ). Acr's have been identified for the type I-D 18 , I-F , II-A and V-A effector complexes (reviewed in 17,19,20 ), numbering over 40 families 21 , but importantly not for type III systems. We focussed on one of the protein families, DUF1874, conserved and widespread ...

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Section: Figure 2 Acriii-1 Rapidly Degrades Ca4 To Linear Products (A)mentioning

confidence: 99%

A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

Athukoralage

McMahon

Zhang

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…For example, a recently discovered Acr is an acetyltransferase that inhibits subtype V-A CRISPR-Cas via acetylation of the effector protein 13 . Another anti-V-A Acr is a nuclease that abrogates the CRISPR-Cas activity by cleaving the guide RNA 14 . Thus, a distinct class of enzymatically active Acrs seems to exist, and at least some of these can be larger and more evolutionarily conserved proteins than the Acrs addressed here.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, recently, enzymatically active Acrs, such as acetyltransferases and nucleases, have been discovered [13][14][15] . Clearly, Acrs have enormous potential for application as modulators of genome editing tools 16,17 .…”

Section: Introductionmentioning

confidence: 99%

Vast diversity of anti-CRISPR proteins predicted with a machine-learning approach

Gussow

Shmakov

Makarova

et al. 2020

Preprint

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Bacteria and archaea evolve under constant pressure from numerous, diverse viruses and thus have evolved multiple defense systems. The CRISPR-Cas are adaptive immunity systems that have been harnessed for the development of the new generation of genome editing and engineering tools. In the incessant host-parasite arms race, viruses evolved multiple anti-defense mechanisms including numerous, diverse anti-CRISPR proteins (Acrs) that can inhibit CRISPR-Cas and therefore have enormous potential for application as modulators of genome editing tools. Most Acrs are small, highly variable proteins which makes their prediction a formidable task. We developed a machine learning approach for comprehensive Acr prediction. The model showed high predictive power when tested against an unseen test set that included several families of recently discovered Acrs and was employed to predict 2,500 novel candidate Acr families. An examination of the top candidates confirms that they possess typical Acr features. One of the top candidates was independently tested and found to possess anti-CRISPR activity (AcrIIA12). We provide a web resource (http://acrcatalog.pythonanywhere.com/) to access the predicted Acrs sequences and annotation. The results of this analysis expand the repertoire of predicted Acrs almost by two orders of magnitude and provide a rich resource for experimental Acr discovery.

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“…They represent a highly diverse class of proteins practically without structural and sequence homology to other proteins (38,39). Acrs inhibit Cas nucleases by various mechanisms, including inhibition of DNA binding (44,45), cleavage of sgRNAs (46), masking catalytic residues and/or inducing Cas9 dimerization (47). AcrIIC3 is an incredibly potent NmeCas9 inhibitor initially discovered in a putative prophage element within the Neisseria meningitidis genome.…”

Section: Introductionmentioning

confidence: 99%

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein

Hoffmann

Mathony

Belzen

et al. 2019

Preprint

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Optogenetic control of CRISPR-Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control their activity via exogenous stimuli persists. The Cas9 from Neisseria meningitidis (Nme) is a particularly small and target-specific Cas9 orthologue, and thus of high interest for in vivo genome editing applications.Here, we report the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein. Building on our previous Acr engineering work, we created hybrids between the NmeCas9 inhibitor AcrIIC3 and the LOV2 blue light sensory domain from Avena sativa. Two AcrIIC3-LOV2 hybrids from our collection potently blocked NmeCas9 activity in the dark, while permitting robust genome editing at various endogenous loci upon blue light irradiation. Structural analysis revealed that, within these hybrids, the LOV2 domain is located in striking proximity to the Cas9 binding surface. Together, our work demonstrates optogenetic regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetic Acrs.

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Broad-spectrum enzymatic inhibition of CRISPR-Cas12a

Cited by 104 publications

References 42 publications

A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

Vast diversity of anti-CRISPR proteins predicted with a machine-learning approach

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein

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