Genetic manipulation of bacteriophages is essential for interrogating phage biology and advancing antimicrobial therapies. However, current genome editing approaches can be inefficient, require multiple steps, or drastically reduce phage titers. Here, we show that targeted DNA nicking enables template-mediated editing of phage genomes in one step without reducing phage titers. Using T7 phage, we show that Cas9-mediated nicking achieved up to 100% recombination across multiple loci, including substitutions and deletions of up to 200 bp and insertions of up to 500 bp, all while preserving phage titers. Editing in T7 was RecA-independent and extended to other phages. Leveraging high titers, we engineered a T7 library of over 440,000 tail-fiber mutants, with isolated mutants restoring infection of two LPS-deficient Escherichia coli hosts by shifting recognition to core LPS components. Overall, DNA nicking is a simple and distinct editing strategy that can advance phage genome engineering, genetic interrogation, and antimicrobial development.

SUMMARY Bacteria encode diverse anti-phage defense systems triggered by invader-specific molecular cues. Here, we report that the compact type III-A Druantia system recognizes exposed single-stranded DNA to drive phage clearance. Using representative systems from Escherichia coli , we show that the two encoded proteins, DruE and DruH, together clear restriction-sensitive or recombination-prone phages without affecting cell growth or viability. DruE dimerizes and engages DNA at exposed single-stranded regions to unwind DNA with 3′-to-5′ directionality, resorting to unique molecular lock, wedge, and clamp elements that aid strand separation and processive translocation. DruH is a monomer in isolation and indirectly interacts with DruE and other host proteins under uninfected conditions, with an infection resulting in the dissociation of the complex. Taken together, our results reveal that exposed single-stranded DNA can trigger bacterial immunity through the directional helicase activity of type III-A Druantia. Highlights The E. coli Druantia III-A defense, comprising DruE and DruH, clears infecting phages DruE dimers bind exposed single-stranded DNA and unwind the upstream DNA duplex 3′-to-5′ DNA unwinding is aided by molecular lock, wedge, and clamp elements DruE interacts with DruH and host proteins, which are displaced upon infection

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated protein (Cas) systems are adaptive immune mechanisms in bacteria and archaea that protect against invading genetic elements by integrating short fragments of foreign DNA into CRISPR arrays. These arrays consist of repetitive sequences interspersed with unique spacers, guiding Cas proteins to recognize and degrade matching nucleic acids. The integrity of these repeat sequences is crucial for the proper function of CRISPR-Cas systems, yet their mutational dynamics remain poorly understood. In this study, we analyzed 56 343 CRISPR arrays across 25 628 diverse prokaryotic genomes to assess the mutation patterns in CRISPR array repeat sequences within and across different CRISPR subtypes. Our findings reveal, as expected to some extent, that mutation frequency is substantially higher in terminal repeat sequences compared to internal repeats consistently across system types. However, the mutation patterns exhibit an unexpected amount of variation among different CRISPR subtypes, suggesting that selective pressures and functional constraints shape repeat sequence evolution in distinct ways. Understanding these mutation dynamics provides insights into the stability and adaptability of CRISPR arrays across diverse bacterial and archaeal lineages. Additionally, we elucidate a novel relationship between repeat mutations and spacer dynamics, demonstrating that hotspots for terminal repeat mutations coincide with regions exhibiting higher spacer conservation. This observation corroborates recent findings indicating that spacer deletions occur at a frequency 374 times greater than that of mutations and are significantly influenced by repeat misalignment. Our findings suggest that repeat mutations might play a pivotal role in spacer retention or loss, or vice versa, thereby highlighting an evolutionary trade-off between the stability and adaptability of CRISPR arrays.

Bacteria and archaea possess an enormous variety of antiviral immune systems that often share homologous proteins and domains. YprA-family helicases are central to widespread defense systems, which include defense island system associated with restriction-modification (DISARM), 7-deazapurine in DNA (Dpd), and Druantia. Through comprehensive phylogenetic and structural analyses of YprA-like helicases, we identify several major clades, which define distinct defense systems including a broad class we call ARMADA (disARM-related antiviral defense array). Apart from the YprA-like helicase, ARMADAs share two more proteins with DISARM, but their YprA homologs are most similar to those of Druantia, which suggests that ARMADA is a missing link connecting DISARM and Druantia. We show experimentally that ARMADA protects bacteria against a broad range of phages via a direct, non-abortive mechanism. We further demonstrate that ARMADA and Druantia Type III systems often co-occur within distinct satellite phage-like mobile elements, which we call SPIDERs (satellite phage integrated defensive and ecotypic replicons) and which provide synergistic resistance against diverse phages.

Many bacteriophages encode anti-CRISPR (Acr) proteins that inhibit bacterial CRISPR-Cas immune systems. Rapid acr gene expression upon phage entry enables CRISPR-Cas neutralization but can impact phage fitness if unregulated. Therefore, Acr production is often controlled by distinct families of co-encoded anti-CRISPR-associated (Aca) proteins, which are usually helix-turn-helix (HTH) regulators that bind DNA within acr-aca operon promoters. Previously, we demonstrated that the Aca2 family additionally represses Acr production translationally by binding structured RNA motifs within the 5' untranslated region (UTR) of the acr-aca mRNA. Here, through systematic bioinformatic analyses, we provide evidence of structured RNA motifs in the 5' UTRs of operons encoding members of other Aca families and show that Aca1 also specifically binds its cognate RNA motif. Additionally, many Aca proteins are predicted to regulate not only their own but also adjacent operons with potential anti-defence genes. Indeed, we show that Aca14, newly identified in this study, represses two predicted anti-defence operons. Aca14 is a ribbon-helix-helix domain protein, revealing regulatory diversity beyond the canonical HTH Aca family members. Collectively, our findings expand our understanding of acr regulation in mobile genetic elements and reveal novel mechanisms by which phages fine-tune anti-defence gene expression.

Selectively eradicating target cells on the basis of their genetic or transcriptional identity remains important in basic research, medicine, biotechnology and agriculture1-3. For applications involving bacteria, CRISPR nucleases offer promising options due to their ability to enact RNA-guided counterselection4-7; however, using these same nucleases for counterselection in eukaryotes has proven much more restrictive8-14. Here we show that Cas12a2, a recently discovered type V CRISPR nuclease, exhibits RNA-triggered DNA shredding15,16, and enables programmable and sequence-specific elimination of yeast and human cells expressing a target transcript. Triggering Cas12a2 elicits rampant double-stranded DNA breaks in trans, leading to cell death. Cell killing can be activated by a wide range of target transcripts, with no observed off-target activation. Leveraging this approach, we selectively eliminate cells that harbour human papillomavirus, cells that failed to undergo gene editing, or cells that encode a prevalent oncogenic point mutation in KRAS. These findings expand the CRISPR toolbox to allow the selective elimination of eukaryotic cells on the basis of their transcriptional profile.

In all domains of life, tRNAs mediate the transfer of genetic information from mRNAs to proteins. As their depletion suppresses translation and, consequently, viral replication, tRNAs represent long-standing and increasingly recognized targets of innate immunity1-5. Here we report Cas12a3 effector nucleases from type V CRISPR-Cas adaptive immune systems in bacteria that preferentially cleave tRNAs after recognition of target RNA. Cas12a3 orthologues belong to one of two previously unreported nuclease clades that exhibit RNA-mediated cleavage of non-target RNA, and are distinct from all other known type V systems. Through cell-based and biochemical assays and direct RNA sequencing, we demonstrate that recognition of a complementary target RNA by the CRISPR RNA triggers Cas12a3 to cleave the conserved 5'-CCA-3' tail of diverse tRNAs to drive growth arrest and anti-phage defence. Cryogenic electron microscopy structures further revealed a distinct tRNA-loading domain that positions the tRNA tail in the RuvC active site of the nuclease. By designing synthetic reporters that mimic the tRNA acceptor stem and tail, we expanded the capacity of current CRISPR-based diagnostics for multiplexed RNA detection. Overall, these findings reveal widespread tRNA inactivation as a previously unrecognized CRISPR-based immune strategy that broadens the application space of the existing CRISPR toolbox.

ABSTRACT Central to CRISPR technologies is the single-guide RNA (sgRNA), an engineered fusion of a processed CRISPR RNA and tracrRNA that directs Cas9 and many Cas12 nucleases to bind and cleave target DNA 1–4 . Here, we report the discovery of similarly compact viral sgRNAs (vsgRNAs) encoded by bacteriophages that counteract bacterial CRISPR-Cas9 immunity. vsgRNAs inhibit Cas9 function via two complementary routes: by sequestering the Cas9 apoenzyme, and by re-directing the Cas9 nuclease to transcriptionally silence its own promoter. vsgRNAs also can cooperate with co-encoded anti-CRISPR proteins (Acrs), including AcrIIA25.1 that blocks DNA binding by Cas9 complexed with a standard sgRNA but not with the vsgRNA. We predict that phages evolved vsgRNAs by co-opting and repurposing host-encoded long-form tracrRNAs (tracr-L) responsible for Cas9 auto-repression and a countermeasure to Acrs 5,6 . Our search also uncovered Cas9-regulating small CRISPR-associated RNAs (scaRNAs), which we predict were inserted upstream of tracrRNAs to form tracr-L but were also co-opted by phages as viral scaRNAs to suppress Cas9 immunity. Finally, we found that vsgRNAs can enable genome editing in mammalian cells, offering a natural guide RNA template for CRISPR technologies. Overall, these findings reveal that bacteriophages devised their own compact sgRNAs tailored to subvert Cas9 immunity, long preceding their rational design to program RNA-guided nucleases 2 .

CRISPR-Cas systems often rely on collateral cleavage of nucleic-acid substrates to combat recognized mobile genetic elements. Of the CRISPR-associated (Cas) RNA-guided effector nucleases, Cas12a2 stands out as the only known example exhibiting rapid collateral cleavage of three distinct substrates: single-stranded (ss)RNA, ssDNA, and double-stranded (ds)DNA, after activating upon binding cognate RNA. However, little is known about the underlying mechanisms of collateral cleavage. Here, we show, using enzyme kinetics and inhibition assays, that Cas12a2 preferentially cleaves collateral DNA over RNA substrates, even when RNA substrates are more abundant. Additionally, using enzyme mutants, enzyme kinetics, and plasmid cleavage assays, we determine that the dsDNA cleavage mechanism relies on the 'aromatic clamp' residues that stabilize unwound and distorted dsDNA in the RuvC nuclease active site. Leveraging the cleavage preference for collateral DNA, we demonstrate that RNA-activated Cas12a2 can readily cleave a ssDNA probe in the presence of high concentrations of non-target RNA, while an RNA-targeting Cas13a cannot. This work provides foundational kinetic and biochemical insights into the collateral cleavage mechanism and substrate preferences of Cas12a2, with immediate implications for understanding Cas12a2-based immunity and developing Cas12a2-based technologies.

CRISPR RNAs (crRNAs) guide recognition and targeting of intracellular invaders as part of adaptive immunity by CRISPR-Cas systems. crRNAs are transcribed from CRISPR arrays of conserved repeats interlaced with invader-derived spacers. While crRNA production is essential for immunity, its optimization for defense remains poorly understood. Here, we show that, in diverse RNA-targeting type VI CRISPR-Cas systems, the leader RNA encoded upstream of the CRISPR array prevents formation of an invader-independent extraneous crRNA (ecrRNA) by blocking processing of the first repeat. Using the VI-B2 system from Porphyromonas gingivalis as a model, we demonstrate that the leader RNA and first repeat form a conserved inhibitory hairpin that precludes binding and processing by the system's Cas13b nuclease. Disrupting this hairpin enables ecrRNA production, which in turn can deplete invader-derived crRNAs and reduce Cas13b-mediated phage defense. Structure prediction indicates that these leader-repeat hairpins are widespread across diverse type VI subtypes, highlighting a conserved regulatory mechanism. Our findings reveal how a prevalent branch of CRISPR-Cas systems suppresses ecrRNA formation to promote RNA-guided immunity.

Natural CRISPR-Cas9 systems rely on crRNA-tracrRNA duplexes to guide DNA targeting. Prior work showed that tracrRNAs could be reprogrammed to hybridize to cellular RNAs, resulting in their conversion into non-canonical crRNAs that enabled RNA detection and recording. However, the fate of the cellular RNA and the engineering opportunities it affords remain unexplored. Here, we show that the hybridized RNA is not inactivated, allowing the recruitment of Cas9 to the RNA duplex to drive RNA base editing and trans-splicing. Fusing ADAR2dd to dSpyCas9 and systematically engineering the reprogrammed tracrRNA (Rptr) enabled efficient and tunable A-to-I RNA editing, with on- and off-target profiles comparable to dCas13. The methodology extended to the compact CjeCas9 that could be further tailored for RNA targeting by deleting the HNH domain and mutating the PAM-interacting domain. Finally, utilizing Rptrs to block splicing enabled 3′ and 5′ RNA trans-splicing. Thus, Rptrs offer a versatile alternative to conventional Cas9 guide RNA architectures for programmable RNA manipulation.

Cas12 nucleases can use guide DNA instead of guide RNA, which switches their targets from DNA to RNA.

Anti-CRISPR proteins (Acrs) inhibit CRISPR-Cas immune defenses, with almost all known Acrs acting on the Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex. Here, we show that AcrVIB1 from Riemerella anatipestifer, the only known Acr against Cas13b, principally acts upstream of RNP complex formation by promoting unproductive crRNA binding followed by crRNA degradation. AcrVIB1 tightly binds to Cas13b but not to the Cas13b-crRNA complex, resulting in enhanced rather than blocked crRNA binding. However, the more tightly bound crRNA does not undergo processing and fails to activate collateral RNA cleavage even with target RNA. The bound crRNA is also accessible to RNases, leading to crRNA turnover in vivo even in the presence of Cas13b. Finally, cryoelectron microscopy (cryo-EM) structures reveal that AcrVIB1 binds a helical domain of Cas13b responsible for securing the crRNA, keeping the domain untethered. These findings reveal an Acr that converts an effector nuclease into a crRNA sink to suppress CRISPR-Cas defense.

The CRISPR-associated (Cas) nuclease Cas12a2 from Sulfuricurvum sp. PC08-66 (SuCas12a2) binds RNA targets with a complementary guide (g)RNA. Target RNA binding causes a major conformational rearrangement in Cas12a2 that activates a RuvC nuclease domain to collaterally cleave RNA, ssDNA and dsDNA, arresting growth and providing population-level immunity. Here, we report in vivo, cell-free, and in vitro methods to characterize the collateral cleavage activity of SuCas12a2 as well as a procedure for gRNA design. As part of the in vivo methods, we describe how to capture growth arrest through plasmid interference and induction of an SOS DNA damage response in the bacterium Escherichia coli. We further apply cell-free transcription-translation to affirm collateral cleavage activity triggered by an expressed RNA target. Finally, as part of the in vitro methods, we describe how to purify active nuclease and subsequently conduct biochemical cleavage assays. In total, the outlined methods should accelerate the exploration of SuCas12a2 and other related Cas nucleases, revealing new features of CRISPR biology and helping develop new CRISPR technologies for molecular diagnostics and other applications.

Cupriavidus necator H16 is a promising microbial platform strain for CO2 valorisation. While C. necator is amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition to industrial applications. In this study, we simplified and accelerated the genome editing pipeline for C. necator by harnessing the Self-splicing Intron-Based Riboswitch (SIBR) system. We used SIBR to tightly control and delay Cas9-based counterselection, achieving >80% editing efficiency at two genomic loci within 48 h after electroporation. To further increase the versatility of the genome editing toolbox, we upgraded SIBR to SIBR2.0 and used it to regulate the expression of Cas12a. SIBR2.0-Cas12a could mediate gene deletion in C. necator with ~70% editing efficiency. Overall, we streamlined the genome editing pipeline for C. necator, facilitating its potential role in the transition to a bio-based economy.

CRISPR-Cas9 systems are widely used for bacterial genome editing, yet their heterologous expression has been associated with cytotoxicity. The Cas9 nuclease from Streptococcus pyogenes (SpyCas9) has been one common source, with reports of cytotoxicity with the nuclease alone or in combination with a single-guide RNA observed in some bacteria. However, the potential cytotoxic effects of other components of the CRISPR-Cas9 system remain unknown. Here, we report that expression of the short isoform of the trans-activating CRISPR RNA (tracr-S) from the S. pyogenes CRISPR-Cas locus is cytotoxic in Lacticaseibacillus paracasei, even in the absence of SpyCas9. Deleting a putative transcription regulator in L. paracasei alleviates tracr-S cytotoxicity and leads to expression of the long isoform of the trans-activating CRISPR RNA (tracr-L). Furthermore, cytotoxicity was specific to the tracr-S sequence and was linked to direct interactions with host RNAs. This work thus reveals that additional CRISPR components beyond Cas9 can interfere with the use of heterologous CRISPR-Cas systems in bacteria, with potential implications for the evolution of CRISPR immunity.

CRISPR–Cas systems are adaptive immune mechanisms in bacteria and archaea that protect against invading genetic elements by integrating short fragments of foreign DNA into CRISPR arrays. These arrays consist of repetitive sequences interspersed with unique spacers, guiding Cas proteins to recognize and degrade matching nucleic acids. The integrity of these repeat sequences is crucial for the proper function of CRISPR–Cas systems, yet their mutational dynamics remain poorly understood. In this study, we analyzed 56,343 CRISPR arrays across 25,628 diverse prokaryotic genomes to assess the mutation patterns in CRISPR array repeat sequences within and across different CRISPR subtypes. Our findings reveal, as expected to some extent, that mutation frequency is substantially higher in terminal repeat sequences compared to internal repeats consistently across system types. However, the mutation patterns exhibit an unexpected amount of variation among different CRISPR subtypes, suggesting that selective pressures and functional constraints shape repeat sequence evolution in distinct ways. Understanding these mutation dynamics provides insights into the stability and adaptability of CRISPR arrays across diverse bacterial and archaeal lineages. Additionally, we elucidate a novel relationship between repeat mutations and spacer dynamics, demonstrating that hotspots for terminal repeat mutations coincide with regions exhibiting spacer conservation. This observation corroborates recent findings by [Fehrenbach et al. (2024)][1] indicating that spacer deletions occur at a frequency 374 times greater than that of mutations and are significantly influenced by repeat misalignment. Our findings suggest that repeat mutations play a pivotal role in spacer retention or loss, or vice versa, thereby highlighting an evolutionary trade-off between the stability and adaptability of CRISPR arrays. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-12

Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here we harness the bacterial antiphage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives homologous recombination, offering flexible and scar-free genome editing without base replacement or counterselection. In tested yeast, plant and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of chemical moieties to DNA to expand current modalities for precision gene editing.

CRISPR-Cas adaptive immune systems in bacteria and archaea enable precise targeting and elimination of invading genetic elements. An inherent feature of these systems is the 'extraneous' CRISPR RNA (ecrRNA), which is produced via the extra repeat in a CRISPR array lacking a corresponding spacer. As ecrRNAs would interact with the Cas machinery yet not direct acquired immunity, they pose a potential barrier to defence. Type II-A CRISPR-Cas systems resolve this barrier through the leader sequence upstream of a CRISPR array, which forms a hairpin structure with the extra repeat that inhibits ecrRNA production. However, the fate of ecrRNAs in other CRISPR types and subtypes remains to be explored. Here, we report that II-C systems likely employ disparate strategies to resolve the ecrRNA due to their distinct configuration in comparison to II-A. Applying bioinformatics analyses to over 650 II-C systems followed by experimental validation, we identified three strategies applicable to these systems: formation of an upstream Rho-independent terminator, formation of a hairpin that sequesters the ecrRNA guide, and mutations in the repeat expected to disrupt ecrRNA formation. These findings expand the list of mechanisms in CRISPR-Cas systems that could resolve the ecrRNA to optimize immune response.

Cas9-SpRY is an engineered variant of the Streptococcus pyogenes Cas9 with relaxed PAM recognition, which can technically be utilized at any target in the genome but some targets are addressed with low efficiency. Here, we show that a previously unexplored motif at the turn and beginning of α-helix 51 (TH51) can be engineered to improve both nuclease and prime-editing activity of Cas9-SpRY. Interaction of the lysine-rich PID loop 2 (PL2) with the target DNA downstream of the PAM (post-PAM) mediates initiation of R-loop formation and subsequent cleavage yet it was unclear if other regions of the PID engage with post-PAM as well. To this end, the NAAN-PAM-targeting iSpyMac hybrid nuclease, which lacks all lysine residues in PL2, was compared with Cas9-SpRY at identical targets using molecular dynamics simulation and in cell culture models, uncovering four crucial post-PAM-interacting lysines in TH51 and TH53 of iSpyMac. Ectopic insertion of a lysine-rich PL2 into iSpyMac boosted its nuclease and prime-editing activities and, in turn, Cas9-SpRY benefited from certain lysine-rich TH51 motifs. Specifically, TH51 from an uncultured Abiotrophia Cas9 species boosted overall Cas9-SpRY activity. Together, this study demonstrates that engineering of post-PAM interacting motifs opens new avenues for the design of advanced CRISPR enzymes.