Activation of Thoeris antiviral system via SIR2 effector filament assembly.

To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems1-7. Some of them (for example, type III CRISPR-Cas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components8-12. In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1''-3' glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD+ (refs. 12-14). Although the structure of ThsA has been solved15, the ThsA activation mechanism remained incompletely understood. Here we show that 1''-3' gcADPR, synthesized in vitro by the dimeric ThsB' protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD+ depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1''-3' gcADPR signal.

Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.

Cas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) effector proteins and show that they provide protection against bacteriophages and plasmids through the targeted DNA binding rather than DNA cleavage. Biochemical assays suggest that Cas12m effectors can act as roadblocks inhibiting DNA transcription and/or replication, thereby triggering interference against invaders. Cryo-EM structure of Gordonia otitidis (Go) Cas12m ternary complex provided here reveals the structural mechanism of DNA binding ensuring interference. Harnessing GoCas12m innate ability to bind DNA target we fused it with adenine deaminase TadA-8e and showed an efficient A-to-G editing in Escherichia coli and human cells. Overall, this study expands our understanding of the functionally diverse Cas12 protein family, revealing DNA-binding dependent interference mechanism of Cas12m effectors that could be harnessed for engineering of compact base-editing tools.

Ribosomal stalk-captured CARF-RelE ribonuclease inhibits translation following CRISPR signaling.

Prokaryotic type III CRISPR-Cas antiviral systems employ cyclic oligoadenylate (cAn) signaling to activate a diverse range of auxiliary proteins that reinforce the CRISPR-Cas defense. Here we characterize a class of cAn-dependent effector proteins named CRISPR-Cas-associated messenger RNA (mRNA) interferase 1 (Cami1) consisting of a CRISPR-associated Rossmann fold sensor domain fused to winged helix-turn-helix and a RelE-family mRNA interferase domain. Upon activation by cyclic tetra-adenylate (cA4), Cami1 cleaves mRNA exposed at the ribosomal A-site thereby depleting mRNA and leading to cell growth arrest. The structures of apo-Cami1 and the ribosome-bound Cami1-cA4 complex delineate the conformational changes that lead to Cami1 activation and the mechanism of Cami1 binding to a bacterial ribosome, revealing unexpected parallels with eukaryotic ribosome-inactivating proteins.

An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.

SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.

The energy landscape for R-loop formation by the CRISPR-Cas Cascade complex.

Clustered regularly interspaced short palindromic repeats (CRISPR) sequences and CRISPR-associated (Cas) genes comprise CIRSPR-Cas effector complexes, which have revolutionized gene editing with their ability to target specific genomic loci using CRISPR RNA (crRNA) complementarity. Recognition of double-stranded DNA targets proceeds via DNA unwinding and base pairing between crRNA and the DNA target strand, forming an R-loop structure. Full R-loop extension is a prerequisite for subsequent DNA cleavage. However, the recognition of unintended sequences with multiple mismatches has limited therapeutic applications and is still poorly understood on a mechanistic level. Here we set up ultrafast DNA unwinding experiments on the basis of plasmonic DNA origami nanorotors to study R-loop formation by the Cascade effector complex in real time, close to base-pair resolution. We resolve a weak global downhill bias of the forming R-loop, followed by a steep uphill bias for the final base pairs. We also show that the energy landscape is modulated by base flips and mismatches. These findings suggest that Cascade-mediated R-loop formation occurs on short timescales in submillisecond single base-pair steps, but on longer timescales in six base-pair intermediate steps, in agreement with the structural periodicity of the crRNA-DNA hybrid.

Dynamic interplay between target search and recognition for a Type I CRISPR-Cas system.

CRISPR-Cas effector complexes enable the defense against foreign nucleic acids and have recently been exploited as molecular tools for precise genome editing at a target locus. To bind and cleave their target, the CRISPR-Cas effectors have to interrogate the entire genome for the presence of a matching sequence. Here we dissect the target search and recognition process of the Type I CRISPR-Cas complex Cascade by simultaneously monitoring DNA binding and R-loop formation by the complex. We directly quantify the effect of DNA supercoiling on the target recognition probability and demonstrate that Cascade uses facilitated diffusion for its target search. We show that target search and target recognition are tightly linked and that DNA supercoiling and limited 1D diffusion need to be considered when understanding target recognition and target search by CRISPR-Cas enzymes and engineering more efficient and precise variants.

TnpB structure reveals minimal functional core of Cas12 nuclease family.

The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells1,2. Bioinformatic analysis identified TnpB proteins as the likely predecessors of Cas12 nucleases3-5, which along with Cas9 are widely used for targeted genome manipulation. Whereas Cas12 family nucleases are well characterized both biochemically and structurally6, the molecular mechanism of TnpB remains unknown. Here we present the cryogenic-electron microscopy structures of the Deinococcus radiodurans TnpB-reRNA (right-end transposon element-derived RNA) complex in DNA-bound and -free forms. The structures reveal the basic architecture of TnpB nuclease and the molecular mechanism for DNA target recognition and cleavage that is supported by biochemical experiments. Collectively, these results demonstrate that TnpB represents the minimal structural and functional core of the Cas12 protein family and provide a framework for developing TnpB-based genome editing tools.

Response regulator PorX coordinates oligonucleotide signalling and gene expression to control the secretion of virulence factors.

The PglZ family of proteins belongs to the alkaline phosphatase superfamily, which consists of metallohydrolases with limited sequence identity but similar metal-coordination architectures in otherwise divergent active sites. Proteins with a well-defined PglZ domain are ubiquitous among prokaryotes as essential components of BREX phage defence systems and two-component systems (TCSs). Whereas other members of the alkaline phosphatase superfamily are well characterized, the activity, structure and biological function of PglZ family proteins remain unclear. We therefore investigated the structure and function of PorX, an orphan response regulator of the Porphyromonas gingivalis TCS containing a putative PglZ effector domain. The crystal structure of PorX revealed a canonical receiver domain, a helical bundle, and an unprecedented PglZ domain, similar to the general organization of the phylogenetically related BREX-PglZ proteins. The PglZ domain of PorX features an active site cleft suitable for large substrates. An extensive search for substrates revealed that PorX is a phosphodiesterase that acts on cyclic and linear oligonucleotides, including signalling molecules such as cyclic oligoadenylates. These results, combined with mutagenesis, biophysical and enzymatic analysis, suggest that PorX coordinates oligonucleotide signalling pathways and indirectly regulates gene expression to control the secretion of virulence factors.

A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex.

CRISPR-Cas effector complexes recognise nucleic acid targets by base pairing with their crRNA which enables easy re-programming of the target specificity in rapidly emerging genome engineering applications. However, undesired recognition of off-targets, that are only partially complementary to the crRNA, occurs frequently and represents a severe limitation of the technique. Off-targeting lacks comprehensive quantitative understanding and prediction. Here, we present a detailed analysis of the target recognition dynamics by the Cascade surveillance complex on a set of mismatched DNA targets using single-molecule supercoiling experiments. We demonstrate that the observed dynamics can be quantitatively modelled as a random walk over the length of the crRNA-DNA hybrid using a minimal set of parameters. The model accurately describes the recognition of targets with single and double mutations providing an important basis for quantitative off-target predictions. Importantly the model intrinsically accounts for observed bias regarding the position and the proximity between mutations and reveals that the seed length for the initiation of target recognition is controlled by DNA supercoiling rather than the Cascade structure.

Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion.

Defence-associated sirtuins (DSRs) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbour an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2-resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defence systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.

Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD+ depletion.

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprising only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here we show that Geobacter sulfurreducens short pAgo and the NAD+-bound Sir2 protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD+ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD+ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD+ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids, underscoring the diversity of mechanisms of prokaryotic Agos.

A new family of CRISPR-type V nucleases with C-rich PAM recognition.

Most CRISPR-type V nucleases are stimulated to cleave double-stranded (ds) DNA targets by a T-rich PAM, which restricts their targeting range. Here, we identify and characterize a new family of type V RNA-guided nuclease, Cas12l, that exclusively recognizes a C-rich (5'-CCY-3') PAM. The organization of genes within its CRISPR locus is similar to type II-B CRISPR-Cas9 systems, but both sequence analysis and functional studies establish it as a new family of type V effector. Biochemical experiments show that Cas12l nucleases function optimally between 37 and 52°C, depending on the ortholog, and preferentially cut supercoiled DNA. Like other type V nucleases, it exhibits collateral nonspecific ssDNA and ssRNA cleavage activity that is triggered by ssDNA or dsDNA target recognition. Finally, we show that one family member, Asp2Cas12l, functions in a heterologous cellular environment, altogether, suggesting that this new group of CRISPR-associated nucleases may be harnessed as genome editing reagents.

Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease.

Transposition has a key role in reshaping genomes of all living organisms1. Insertion sequences of IS200/IS605 and IS607 families2 are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established3-5. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR-Cas9/Cas12 nucleases6-8. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5'-TTGAT transposon-associated motif. We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR-Cas nucleases and establishes TnpB as a prototype of a new system for genome editing.

Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells.

Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools.

Discrimination of single-point mutations in unamplified genomic DNA via Cas9 immobilized on a graphene field-effect transistor.

Simple and fast methods for the detection of target genes with single-nucleotide specificity could open up genetic research and diagnostics beyond laboratory settings. We recently reported a biosensor for the electronic detection of unamplified target genes using liquid-gated graphene field-effect transistors employing an RNA-guided catalytically deactivated CRISPR-associated protein 9 (Cas9) anchored to a graphene monolayer. Here, using unamplified genomic samples from patients and by measuring multiple types of electrical response, we show that the biosensors can discriminate within one hour between wild-type and homozygous mutant alleles differing by a single nucleotide. We also show that biosensors using a guide RNA-Cas9 orthologue complex targeting genes within the protospacer-adjacent motif discriminated between homozygous and heterozygous DNA samples from patients with sickle cell disease, and that the biosensors can also be used to rapidly screen for guide RNA-Cas9 complexes that maximize gene-targeting efficiency.

HEPN-MNT Toxin-Antitoxin System: The HEPN Ribonuclease Is Neutralized by OligoAMPylation.

Prokaryotic toxin-antitoxin (TA) systems are composed of a toxin capable of interfering with key cellular processes and its neutralizing antidote, the antitoxin. Here, we focus on the HEPN-MNT TA system encoded in the vicinity of a subtype I-D CRISPR-Cas system in the cyanobacterium Aphanizomenon flos-aquae. We show that HEPN acts as a toxic RNase, which cleaves off 4 nt from the 3' end in a subset of tRNAs, thereby interfering with translation. Surprisingly, we find that the MNT (minimal nucleotidyltransferase) antitoxin inhibits HEPN RNase through covalent di-AMPylation (diadenylylation) of a conserved tyrosine residue, Y109, in the active site loop. Furthermore, we present crystallographic snapshots of the di-AMPylation reaction at different stages that explain the mechanism of HEPN RNase inactivation. Finally, we propose that the HEPN-MNT system functions as a cellular ATP sensor that monitors ATP homeostasis and, at low ATP levels, releases active HEPN toxin.

A catalogue of biochemically diverse CRISPR-Cas9 orthologs.

Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains.

The type III CRISPR-Cas systems provide immunity against invading nucleic acids through the coordinated transcription-dependent DNA targeting and cyclic adenylate (cAn)-activated RNA degradation. Here, we show that both these pathways contribute to the Streptococcus thermophilus (St) type III-A CRISPR-Cas immunity. HPLC-MS analysis revealed that in the heterologous Escherichia coli host the StCsm effector complex predominantly produces cA5 and cA6. cA6 acts as a signaling molecule that binds to the CARF domain of StCsm6 to activate non-specific RNA degradation by the HEPN domain. By dissecting StCsm6 domains we demonstrate that both CARF and HEPN domains act as ring nucleases that degrade cAns to switch signaling off. CARF ring nuclease converts cA6 to linear A6>p and to the final A3>p product. HEPN domain, which typically degrades RNA, also shows ring nuclease activity and indiscriminately degrades cA6 or other cAns down to A>p. We propose that concerted action of both ring nucleases enables self-regulation of the RNase activity in the HEPN domain and eliminates all cAn secondary messengers in the cell when viral infection is combated by a coordinated action of Csm effector and the cA6-activated Csm6 ribonuclease.