RESUMEN
CasDinG is an ATP-dependent 5'-3' DNA helicase essential for bacterial Type IV-A1 CRISPR associated immunity. CasDinG contains an essential N-terminal domain predicted to bind DNA. To better understand the role of the N-terminal domain, we attempted to co-crystallize CasDinG with DNA substrates. We successfully crystallized CasDinG in a tightly packed, crystal conformation with previously unobserved unit cell dimensions. However, the structure lacked electron density for a bound DNA substrate and the CasDinG N-terminal domain. Additionally, the tight crystal packing disallowed space for the N-terminal domain, indicating that the N-terminal domain was proteolyzed before crystallization. Follow up experiments revealed that the N-terminal domain of CasDinG is proteolyzed after a few days at room temperature, but is protected from proteolysis at 4°C. These data provide a distinct x-ray crystal structure of CasDinG and indicate the essential N-terminal domain of CasDinG is prone to proteolysis.
RESUMEN
CRISPR-associated DinG protein (CasDinG) is essential to type IV-A CRISPR function. Here, we demonstrate that CasDinG from Pseudomonas aeruginosa strain 83 is an ATP-dependent 5'-3' DNA translocase that unwinds double-stranded (ds)DNA and RNA/DNA hybrids. The crystal structure of CasDinG reveals a superfamily 2 helicase core of two RecA-like domains with three accessory domains (N-terminal, arch, and vestigial FeS). To examine the in vivo function of these domains, we identified the preferred PAM sequence for the type IV-A system (5'-GNAWN-3' on the 5'-side of the target) with a plasmid library and performed plasmid clearance assays with domain deletion mutants. Plasmid clearance assays demonstrated that all three domains are essential for type IV-A immunity. Protein expression and biochemical assays suggested the vFeS domain is needed for protein stability and the arch for helicase activity. However, deletion of the N-terminal domain did not impair ATPase, ssDNA binding, or helicase activities, indicating a role distinct from canonical helicase activities that structure prediction tools suggest involves interaction with dsDNA. This work demonstrates CasDinG helicase activity is essential for type IV-A CRISPR immunity as well as the yet undetermined activity of the CasDinG N-terminal domain.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN de Cadena Simple , ADN de Cadena Simple/genética , ADN Helicasas/metabolismo , ADN/genética , ARN Helicasas/genética , ARNRESUMEN
Bacterial abortive-infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate1,2. Several RNA-targeting CRISPR-Cas systems (that is, types III and VI) cause abortive-infection phenotypes by activating indiscriminate nucleases3-5. However, a CRISPR-mediated abortive mechanism that leverages indiscriminate DNase activity of an RNA-guided single-effector nuclease has yet to be observed. Here we report that RNA targeting by the type V single-effector nuclease Cas12a2 drives abortive infection through non-specific cleavage of double-stranded DNA (dsDNA). After recognizing an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and dsDNA. Within cells, the activation of Cas12a2 induces an SOS DNA-damage response and impairs growth, preventing the dissemination of the invader. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided RNA-targeting tool. These findings expand the known defensive abilities of CRISPR-Cas systems and create additional opportunities for CRISPR technologies.
Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , ADN , ARN , Proteínas Asociadas a CRISPR/metabolismo , ADN/metabolismo , ADN de Cadena Simple/metabolismo , ARN/metabolismo , Respuesta SOS en Genética , Daño del ADN , ARN Guía de Sistemas CRISPR-Cas , Edición GénicaRESUMEN
Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a complementary RNA target, culminating in abortive infection1. Here we report structures of Cas12a2 in binary, ternary and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of 'aromatic clamp' residues that are crucial for double-stranded DNA degradation and in vivo immune system function. Our work provides a structural basis for this mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.
Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , ARN , Proteínas Asociadas a CRISPR/antagonistas & inhibidores , Proteínas Asociadas a CRISPR/metabolismo , ADN/química , ADN/inmunología , ADN/metabolismo , ARN/química , ARN/metabolismo , Activación Enzimática , Dominio Catalítico , Especificidad por SustratoRESUMEN
Type IV CRISPR systems encode CRISPR associated (Cas)-like proteins that combine with small RNAs to form multi-subunit ribonucleoprotein complexes. However, the lack of Cas nucleases, integrases, and other genetic features commonly observed in most CRISPR systems has made it difficult to predict type IV mechanisms of action and biological function. Here we summarize recent bioinformatic and experimental advancements that collectively provide the first glimpses into the function of specific type IV subtypes. We also provide a bioinformatic and structural analysis of type IV-specific proteins within the context of multi-subunit (class 1) CRISPR systems, informing future studies aimed at elucidating the function of these cryptic systems.
RESUMEN
We reveal the cryo-electron microscopy structure of a type IV-B CRISPR ribonucleoprotein (RNP) complex (Csf) at 3.9-Å resolution. The complex best resembles the type III-A CRISPR Csm effector complex, consisting of a Cas7-like (Csf2) filament intertwined with a small subunit (Cas11) filament, but the complex lacks subunits for RNA processing and target DNA cleavage. Surprisingly, instead of assembling around a CRISPR-derived RNA (crRNA), the complex assembles upon heterogeneous RNA of a regular length arranged in a pseudo-A-form configuration. These findings provide a high-resolution glimpse into the assembly and function of enigmatic type IV CRISPR systems, expanding our understanding of class I CRISPR-Cas system architecture, and suggesting a function for type IV-B RNPs that may be distinct from other class 1 CRISPR-associated systems.
RESUMEN
Bacteria and archaea use CRISPR-Cas adaptive immune systems to destroy complementary nucleic acids using RNAs derived from CRISPR loci. Here, we provide the first functional evidence for type IV CRISPR-Cas, demonstrating that the system from Pseudomonas aeruginosa strain PA83 mediates RNA-guided interference against a plasmid in vivo, both clearing the plasmid and inhibiting its uptake. This interference depends on the putative NTP-dependent helicase activity of Csf4/DinG.
Asunto(s)
Sistemas CRISPR-Cas/genética , Pseudomonas aeruginosa/genética , ARN Guía de Kinetoplastida/genética , Bacterias/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Plásmidos/genética , Interferencia de ARN/fisiologíaRESUMEN
Prokaryotic CRISPR-Cas adaptive immune systems rely on small non-coding RNAs derived from CRISPR loci to recognize and destroy complementary nucleic acids. However, the mechanism of Type IV CRISPR RNA (crRNA) biogenesis is poorly understood. To dissect the mechanism of Type IV CRISPR RNA biogenesis, we determined the x-ray crystal structure of the putative Type IV CRISPR associated endoribonuclease Cas6 from Mahella australiensis (Ma Cas6-IV) and characterized its enzymatic activity with RNA cleavage assays. We show that Ma Cas6-IV specifically cleaves Type IV crRNA repeats at the 3' side of a predicted stem loop, with a metal-independent, single-turnover mechanism that relies on a histidine and a tyrosine located within the putative endonuclease active site. Structure and sequence alignments with Cas6 orthologs reveal that although Ma Cas6-IV shares little sequence homology with other Cas6 proteins, all share common structural features that bind distinct crRNA repeat sequences. This analysis of Type IV crRNA biogenesis provides a structural and biochemical framework for understanding the similarities and differences of crRNA biogenesis across multi-subunit Class 1 CRISPR immune systems.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ARN/química , ARN/genética , Transcripción Genética , Secuencia de Aminoácidos , Proteínas Asociadas a CRISPR/química , Catálisis , Dominio Catalítico , Firmicutes/genética , Firmicutes/metabolismo , Orden Génico , Modelos Moleculares , Conformación Molecular , Conformación de Ácido Nucleico , Precursores del ARN , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Prokaryotes use primed CRISPR adaptation to update their memory bank of spacers against invading genetic elements that have escaped CRISPR interference through mutations in their protospacer target site. We previously observed a trend that nucleotide-dependent mismatches between crRNA and the protospacer strongly influence the efficiency of primed CRISPR adaptation. Here we show that guanine-substitutions in the target strand of the protospacer are highly detrimental to CRISPR interference and interference-dependent priming, while cytosine-substitutions are more readily tolerated. Furthermore, we show that this effect is based on strongly decreased binding affinity of the effector complex Cascade for guanine-mismatched targets, while cytosine-mismatched targets only minimally affect target DNA binding. Structural modeling of Cascade-bound targets with mismatches shows that steric clashes of mismatched guanines lead to unfavorable conformations of the RNA-DNA duplex. This effect has strong implications for the natural selection of target site mutations that lead to effective escape from type I CRISPR-Cas systems.
Asunto(s)
Sistemas CRISPR-Cas , Citosina/química , Escherichia coli/genética , Guanina/química , ARN Bacteriano/genética , ARN Guía de Kinetoplastida/genética , Emparejamiento Base , Secuencia de Bases , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Citosina/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Guanina/metabolismo , Mutación , Plásmidos/química , Plásmidos/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/metabolismoRESUMEN
Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here, we use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Complejos Multiproteicos/metabolismo , ADN/química , ADN de Cadena Simple/química , Proteínas de Unión al ADN/genética , Deuterio , Escherichia coli/enzimología , Espectrometría de Masas , Complejos Multiproteicos/genética , Mutación , Conformación de Ácido Nucleico , Unión ProteicaRESUMEN
Adaptive immune systems in bacteria and archaea rely on small CRISPR-derived RNAs (crRNAs) to guide specialized nucleases to foreign nucleic acids. The activation of these nucleases is controlled by a series of molecular checkpoints that ensure precise cleavage of nucleic acid targets, while minimizing toxic off-target cleavage events. In this review, we highlight recent advances in understanding regulatory mechanisms responsible for controlling the activation of these nucleases and identify emerging regulatory themes conserved across diverse CRISPR systems.
Asunto(s)
Regulación Alostérica , Sistemas CRISPR-Cas , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Archaea/enzimología , Bacterias/enzimología , Modelos BiológicosRESUMEN
Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR-mediated adaptive immune systems for protection from viral infection, and viruses have evolved diverse anti-CRISPR (Acr) proteins that subvert these immune systems. The adaptive immune system in Pseudomonas aeruginosa (type I-F) relies on a 350 kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation. Here, we report the cryo-electron microscopy (cryo-EM) structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3.4 Å. The structure explains the molecular mechanism for immune system suppression, and structure-guided mutations show that the Acr proteins bind to residues essential for crRNA-mediated detection of DNA. Collectively, these data provide a snapshot of an ongoing molecular arms race between viral suppressors and the immune system they target.
Asunto(s)
Bacteriófagos/química , Proteínas Asociadas a CRISPR/química , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/virología , ARN Bacteriano/química , Proteínas Virales/química , Bacteriófagos/clasificación , Bacteriófagos/genética , Microscopía por Crioelectrón , Cristalografía por Rayos X , Vigilancia Inmunológica , Modelos Moleculares , Pseudomonas aeruginosa/genética , ARN Bacteriano/metabolismo , ARN Bacteriano/ultraestructura , Proteínas Virales/ultraestructuraRESUMEN
Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of this complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Finally, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.
Asunto(s)
Sistemas CRISPR-Cas/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Sustancias Macromoleculares/metabolismo , ARN Bacteriano/genética , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , Proteínas Asociadas a CRISPR/metabolismo , ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Silenciador del Gen , Sustancias Macromoleculares/química , ARN Bacteriano/química , Transcripción GenéticaRESUMEN
In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.
Asunto(s)
Proteínas Asociadas a CRISPR/química , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Arginina/química , Proteínas Asociadas a CRISPR/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Lisina/química , Modelos Moleculares , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Unión Proteica , Conformación Proteica , ARN Bacteriano/metabolismoRESUMEN
Structures of multisubunit macromolecular machines are primarily determined either by electron microscopy (EM) or by X-ray crystallography. In many cases, a structure for a complex can be obtained at low resolution (at a coarse level of detail) with EM and at a higher resolution (with finer detail) by X-ray crystallography. The integration of these two structural techniques is becoming increasingly important for the generation of atomic models of macromolecular complexes. A low-resolution EM image can be a powerful tool for obtaining the 'phase' information that is missing from an X-ray crystallography experiment; however, integration of EM and X-ray diffraction data has been technically challenging. Here we present a step-by-step protocol that explains how low-resolution EM maps can be placed in the crystallographic unit cell by molecular replacement, and how initial phases computed from the placed EM density are extended to high resolution by averaging maps over noncrystallographic symmetry. As the resolution gap between EM and X-ray crystallography continues to narrow, the use of EM maps to help with X-ray crystal structure determination, as described in this protocol, will become increasingly effective.
Asunto(s)
Cristalografía por Rayos X , Microscopía Electrónica , Estructura Molecular , Proteínas Asociadas a CRISPR , Escherichia coliRESUMEN
Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for targeted elimination of foreign nucleic acids. These immune systems have been divided into three main types, and the first atomic-resolution structure of a type III RNA-guided immune complex provides new insights into the mechanisms of nucleic acid degradation. Here we compare the crystal structure of a type III complex to recently determined structures of DNA-targeting type I CRISPR complexes. Structural comparisons support previous assertions that type I and type III systems share a common ancestor and reveal how a conserved structural chassis is used to support RNA-, DNA-, or both RNA- and DNA-targeting mechanisms.
Asunto(s)
Archaea/virología , Bacterias/virología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas Asociadas a CRISPR/química , Modelos Moleculares , Filogenia , Unión Proteica , Conformación Proteica , ARN de Archaea/química , ARN de Archaea/fisiología , ARN Bacteriano/química , ARN Bacteriano/fisiologíaRESUMEN
Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3'-5' turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The 'ratchet helix' is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates.
Asunto(s)
ARN Helicasas DEAD-box/química , ARN/química , Proteínas de Saccharomyces cerevisiae/química , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Modelos Moleculares , Mutación , Poli A/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of RNA-guided adaptive immune systems that protect bacteria and archaea from viruses and plasmids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Here we present the 3.24 angstrom resolution x-ray crystal structure of Cascade. Eleven proteins and a 61-nucleotide crRNA assemble into a seahorse-shaped architecture that binds double-stranded DNA targets complementary to the crRNA-guide sequence. Conserved sequences on the 3' and 5' ends of the crRNA are anchored by proteins at opposite ends of the complex, whereas the guide sequence is displayed along a helical assembly of six interwoven subunits that present five-nucleotide segments of the crRNA in pseudo-A-form configuration. The structure of Cascade suggests a mechanism for assembly and provides insights into the mechanisms of target recognition.
Asunto(s)
Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas de Escherichia coli/química , Escherichia coli/genética , ARN Bacteriano/química , Cristalografía por Rayos X , Edición de ARN , ARN Pequeño no TraducidoRESUMEN
Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR-Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea.
Asunto(s)
Bacterias/inmunología , Sistemas CRISPR-Cas/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Archaea/genética , Archaea/inmunología , Bacterias/genética , Sistemas CRISPR-Cas/genética , Modelos Moleculares , Plásmidos/genética , ARN Bacteriano/fisiología , Ribonucleoproteínas/química , Ribonucleoproteínas/fisiología , Virus/genéticaRESUMEN
Helicases utilize NTPs to modulate their binding to nucleic acids and many of these enzymes also unwind DNA or RNA duplexes in an NTP-dependent fashion. These proteins are phylogenetically related but functionally diverse, with essential roles in virtually all aspects of nucleic acid metabolism. A new class of helicases associated with RNA-guided adaptive immune systems in bacteria and archaea has recently been identified. Prokaryotes acquire resistance to invading genetic parasites by integrating short fragments of foreign nucleic acids into repetitive loci in the host chromosome known as CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR-associated gene 3 (cas3) encodes a conserved helicase protein that is essential for phage defense. Here we review recent advances in Cas3 biology, and provide a new phylogenetic framework that positions Cas3 in the helicase family tree. We anticipate that this Cas3 phylogeny will guide future biochemical and structural studies.