Search

Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. Recently, a highly precise subtype I-F1 CRISPR-Cas system (HNH-Cascade) was found that lacks Cas3, the absence of which is compensated for by the insertion of an HNH endonuclease domain in the Cas8 Cascade component. Here, we describe the cryo-EM structure of Selenomonas sp. HNH-Cascade (SsCascade) in complex with target DNA and characterize its mechanism of action. The Cascade scaffold is complemented by the HNH domain, creating a ring-like structure in which the unwound target DNA is precisely cleaved. This structure visualizes a unique hybrid of two extensible biological systems-Cascade, an evolutionary platform for programmable DNA effectors, and an HNH nuclease, an adaptive domain with a spectrum of enzymatic activity., Competing Interests: Declaration of interests S.H., H.A.-T., and F.Z. are coinventors on a patent application (PCT/US2023/070150) related to this work filed by the Broad Institute and MIT. F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, Aera Therapeutics, and Moonwalk Biosciences. F.Z. is a scientific advisor for Octant., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The TnpB proteins are transposon-associated RNA-guided nucleases that are among the most abundant proteins encoded in bacterial and archaeal genomes, but whose functions in the transposon life cycle remain unknown. TnpB appears to be the evolutionary ancestor of Cas12, the effector nuclease of type V CRISPR-Cas systems. We performed a comprehensive census of TnpBs in archaeal and bacterial genomes and constructed a phylogenetic tree on which we mapped various features of these proteins. In multiple branches of the tree, the catalytic site of the TnpB nuclease is rearranged, demonstrating structural and probably biochemical malleability of this enzyme. We identified numerous cases of apparent recruitment of TnpB for other functions of which the most common is the evolution of type V CRISPR-Cas effectors on about 50 independent occasions. In many other cases of more radical exaptation, the catalytic site of the TnpB nuclease is apparently inactivated, suggesting a regulatory function, whereas in others, the activity appears to be retained, indicating that the recruited TnpB functions as a nuclease, for example, as a toxin. These findings demonstrate remarkable evolutionary malleability of the TnpB scaffold and provide extensive opportunities for further exploration of RNA-guided biological systems as well as multiple applications., Competing Interests: Competing interests statement:F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, and Aera Therapeutics. F.Z. is also a scientific advisor for Octant.

Microbial systems underpin many biotechnologies, including CRISPR, but the exponential growth of sequence databases makes it difficult to find previously unidentified systems. In this work, we develop the fast locality-sensitive hashing-based clustering (FLSHclust) algorithm, which performs deep clustering on massive datasets in linearithmic time. We incorporated FLSHclust into a CRISPR discovery pipeline and identified 188 previously unreported CRISPR-linked gene modules, revealing many additional biochemical functions coupled to adaptive immunity. We experimentally characterized three HNH nuclease-containing CRISPR systems, including the first type IV system with a specified interference mechanism, and engineered them for genome editing. We also identified and characterized a candidate type VII system, which we show acts on RNA. This work opens new avenues for harnessing CRISPR and for the broader exploration of the vast functional diversity of microbial proteins.

RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR-Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors such as Cas9 and Cas12 perform guide-RNA-dependent DNA cleavage 1 . Although a few eukaryotic RNA-guided systems have been studied, including RNA interference 2 and ribosomal RNA modification 3 , it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided systems (termed OMEGA) was reported 4,5 . The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity 4,6 . TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins 4,7 , raising the possibility that eukaryotes are also equipped with CRISPR-Cas or OMEGA-like programmable RNA-guided endonucleases. Here we report the biochemical characterization of Fz, showing that it is an RNA-guided DNA endonuclease. We also show that Fz can be reprogrammed for human genome engineering applications. Finally, we resolve the structure of Spizellomyces punctatus Fz at 2.7 Å using cryogenic electron microscopy, showing the conservation of core regions among Fz, TnpB and Cas12, despite diverse cognate RNA structures. Our results show that Fz is a eukaryotic OMEGA system, demonstrating that RNA-guided endonucleases are present in all three domains of life., (© 2023. The Author(s).)

To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously uncharacterized target-selector proteins found in newly discovered transposable elements (TEs). We experimentally characterized a CAST I-D system and a Tn6022-like transposon that uses TnsF, which contains an inactivated tyrosine recombinase domain, to target the comM gene. Additionally, we identified a non-Tn7 transposon, Tsy, encoding a homolog of TnsF with an active tyrosine recombinase domain, which we show also inserts into comM. Our findings show that Tn7 transposons employ modular architecture and co-opt target selectors from various sources to optimize target selection and drive transposon spread., Competing Interests: Declaration of interests F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, Proof Diagnostics, and Aera Therapeutics. F.Z. is a scientific advisor for Octant. G.F., M.K., and F.Z. are co-inventors on a provisional patent application filed by Broad relating to this work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

TnpB is a member of the Obligate Mobile Element Guided Activity (OMEGA) RNA-guided nuclease family, is harbored in transposons, and likely functions to maintain the transposon in genomes. Previously, it was shown that TnpB cleaves double- and single-stranded DNA substrates in an RNA-guided manner, but the biogenesis of the TnpB ribonucleoprotein (RNP) complex is unknown. Using in vitro purified apo TnpB, we demonstrate the ability of TnpB to generate guide omegaRNA (ωRNA) from its own mRNA through 5' processing. We also uncover a potential cis -regulatory mechanism whereby a region of the TnpB mRNA inhibits DNA cleavage by the RNP complex. We further expand the characterization of TnpB by examining ωRNA processing and RNA-guided nuclease activity in 59 orthologs spanning the natural diversity of the TnpB family. This work reveals a new functionality, ωRNA biogenesis, of TnpB, and characterizes additional members of this biotechnologically useful family of programmable enzymes.

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins 1 . Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases 2,3 . TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors., (© 2023. The Author(s).)

Transposon-encoded IscB family proteins are RNA-guided nucleases in the OMEGA (obligate mobile element-guided activity) system, and likely ancestors of the RNA-guided nuclease Cas9 in the type II CRISPR-Cas adaptive immune system. IscB associates with its cognate ωRNA to form a ribonucleoprotein complex that cleaves double-stranded DNA targets complementary to an ωRNA guide segment. Although IscB shares the RuvC and HNH endonuclease domains with Cas9, it is much smaller than Cas9, mainly due to the lack of the α-helical nucleic-acid recognition lobe. Here, we report the cryo-electron microscopy structure of an IscB protein from the human gut metagenome (OgeuIscB) in complex with its cognate ωRNA and a target DNA, at 2.6-Å resolution. This high-resolution structure reveals the detailed architecture of the IscB-ωRNA ribonucleoprotein complex, and shows how the small IscB protein assembles with the ωRNA and mediates RNA-guided DNA cleavage. The large ωRNA scaffold structurally and functionally compensates for the recognition lobe of Cas9, and participates in the recognition of the guide RNA-target DNA heteroduplex. These findings provide insights into the mechanism of the programmable DNA cleavage by the IscB-ωRNA complex and the evolution of the type II CRISPR-Cas9 effector complexes., (© 2022. The Author(s).)

RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies 1,2 . Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development 3 . Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely ancestor of Cas9, and the nickase IsrB, a homologue of IscB lacking the HNH nuclease domain 4 . IsrB consists of only around 350 amino acids, but its small size is counterbalanced by a relatively large RNA guide (roughly 300-nt ωRNA). Here, we report the cryogenic-electron microscopy structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with its cognate ωRNA and a target DNA. We find the overall structure of the IsrB protein shares a common scaffold with Cas9. In contrast to Cas9, however, which uses a recognition (REC) lobe to facilitate target selection, IsrB relies on its ωRNA, part of which forms an intricate ternary structure positioned analogously to REC. Structural analyses of IsrB and its ωRNA as well as comparisons to other RNA-guided systems highlight the functional interplay between protein and RNA, advancing our understanding of the biology and evolution of these diverse systems., (© 2022. The Author(s).)

Type VI CRISPR-Cas13 effector enzymes catalyze RNA-guided RNA cleavage and have been harnessed for various technologies, such as RNA detection, targeting, and editing. Recent studies identified Cas13bt3 (also known as Cas13X.1) as a miniature Cas13 enzyme, which can be used for knockdown and editing of target transcripts in mammalian cells. However, the action mechanism of the compact Cas13bt3 remains unknown. Here, we report the structures of the Cas13bt3-guide RNA complex and the Cas13bt3-guide RNA-target RNA complex. The structures revealed how Cas13bt3 recognizes the guide RNA and its target RNA and provided insights into the activation mechanism of Cas13bt3, which is distinct from those of the other Cas13a/d enzymes. Furthermore, we rationally engineered enhanced Cas13bt3 variants and ultracompact RNA base editors. Overall, this study improves our mechanistic understanding of the CRISPR-Cas13 enzymes and paves the way for the development of efficient Cas13-mediated transcriptome modulation technologies., Competing Interests: Declaration of interests F.Z. is a co-founder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, and Sherlock Biosciences. O.N. is a co-founder, board member, and scientific advisor of Curreio. R.N., S.K., H.A.-T., F.Z., H.N., and O.N. have filed a patent application related to this work., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

CRISPR-Cas13 systems have been developed for precise RNA editing, and can potentially be used therapeutically when temporary changes are desirable or when DNA editing is challenging. We have identified and characterized an ultrasmall family of Cas13b proteins-Cas13bt-that can mediate mammalian transcript knockdown. We have engineered compact variants of REPAIR and RESCUE RNA editors by functionalizing Cas13bt with adenosine and cytosine deaminase domains, and demonstrated packaging of the editors within a single adeno-associated virus., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Transposition is a major mechanism of horizontal gene mobility in prokaryotes. However, exploration of the genes mobilized by transposons (cargo) is hampered by the difficulty in delineating integrated transposons from their surrounding genetic context. Here, we present a computational approach that allowed us to identify the boundaries of 6,549 Tn 7 -like transposons. We found that 96% of these transposons carry at least one cargo gene. Delineation of distinct communities in a gene-sharing network demonstrates how transposons function as a conduit of genes between phylogenetically distant hosts. Comparative analysis of the cargo genes reveals significant enrichment of mobile genetic elements (MGEs) nested within Tn 7 -like transposons, such as insertion sequences and toxin-antitoxin modules, and of genes involved in recombination, anti-MGE defense, and antibiotic resistance. More unexpectedly, cargo also includes genes encoding central carbon metabolism enzymes. Twenty-two Tn 7 -like transposons carry both an anti-MGE defense system and antibiotic resistance genes, illustrating how bacteria can overcome these combined pressures upon acquisition of a single transposon. This work substantially expands the distribution of Tn 7 -like transposons, defines their evolutionary relationships, and provides a large-scale functional classification of prokaryotic genes mobilized by transposition. IMPORTANCE Transposons are major vehicles of horizontal gene transfer that, in addition to genes directly involved in transposition, carry cargo genes. However, characterization of these genes is hampered by the difficulty of identification of transposon boundaries. We developed a computational approach for detecting transposon ends and applied it to perform a comprehensive census of the cargo genes of Tn 7 -like transposons, a large class of bacterial mobile genetic elements (MGE), many of which employ a unique, CRISPR-mediated mechanism of site-specific transposition. The cargo genes encompass a striking diversity of MGE, defense, and antibiotic resistance systems. Unexpectedly, we also identified cargo genes encoding metabolic enzymes. Thus, Tn 7 -like transposons mobilize a vast repertoire of genes that can have multiple effects on the host bacteria.

IscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interactions with any RNA remain uncharacterized. Using evolutionary analysis, RNA sequencing, and biochemical experiments, we reconstructed the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons. We found that IscB uses a single noncoding RNA for RNA-guided cleavage of double-stranded DNA and can be harnessed for genome editing in human cells. We also demonstrate the RNA-guided nuclease activity of TnpB, another IS200/IS605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases. This work reveals a widespread class of transposon-encoded RNA-guided nucleases, which we name OMEGA (obligate mobile element–guided activity), with strong potential for developing as biotechnologies.

Tn7-like transposons have co-opted CRISPR systems, including class 1 type I-F, I-B, and class 2 type V-K. Intriguingly, although these CRISPR-associated transposases (CASTs) undergo robust CRISPR RNA (crRNA)-guided transposition, they are almost never found in sites targeted by the crRNAs encoded by the cognate CRISPR array. To understand this paradox, we investigated CAST V-K and I-B systems and found two distinct modes of transposition: (1) crRNA-guided transposition and (2) CRISPR array-independent homing. We show distinct CAST systems utilize different molecular mechanisms to target their homing site. Type V-K CAST systems use a short, delocalized crRNA for RNA-guided homing, whereas type I-B CAST systems, which contain two distinct target selector proteins, use TniQ for RNA-guided DNA transposition and TnsD for homing to an attachment site. These observations illuminate a key step in the life cycle of CAST systems and highlight the diversity of molecular mechanisms mediating transposon homing., Competing Interests: Declaration of interests The Broad Institute has filed patent applications related to this work. F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, and Sherlock Biosciences., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Repetitive sequence elements in proteins and nucleic acids are often signatures of adaptive or reprogrammable systems in nature. Known examples of these systems, such as transcriptional activator-like effectors (TALE) and CRISPR, have been harnessed as powerful molecular tools with a wide range of applications including genome editing. The continued expansion of genomic sequence databases raises the possibility of prospectively identifying new such systems by computational mining. By leveraging sequence repeats as an organizing principle, here we develop a systematic genome mining approach to explore new types of naturally adaptive systems, five of which are discussed in greater detail. These results highlight the existence of a diverse range of intriguing systems in nature that remain to be explored and also provide a framework for future discovery efforts., Competing Interests: F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, and Sherlock Biosciences., (© The Author(s) 2020. Published by Cambridge University Press 2020.)

Despite the widespread implementation of public health measures, coronavirus disease 2019 (COVID-19) continues to spread in the United States. To facilitate an agile response to the pandemic, we developed How We Feel, a web and mobile application that collects longitudinal self-reported survey responses on health, behaviour and demographics. Here, we report results from over 500,000 users in the United States from 2 April 2020 to 12 May 2020. We show that self-reported surveys can be used to build predictive models to identify likely COVID-19-positive individuals. We find evidence among our users for asymptomatic or presymptomatic presentation; show a variety of exposure, occupational and demographic risk factors for COVID-19 beyond symptoms; reveal factors for which users have been SARS-CoV-2 PCR tested; and highlight the temporal dynamics of symptoms and self-isolation behaviour. These results highlight the utility of collecting a diverse set of symptomatic, demographic, exposure and behavioural self-reported data to fight the COVID-19 pandemic.

Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Recent advances in machine learning have made significant contributions to drug discovery. Deep neural networks in particular have been demonstrated to provide significant boosts in predictive power when inferring the properties and activities of small-molecule compounds (Ma, J. et al. J. Chem. Inf., Model: 2015, 55, 263-274). However, the applicability of these techniques has been limited by the requirement for large amounts of training data. In this work, we demonstrate how one-shot learning can be used to significantly lower the amounts of data required to make meaningful predictions in drug discovery applications. We introduce a new architecture, the iterative refinement long short-term memory, that, when combined with graph convolutional neural networks, significantly improves learning of meaningful distance metrics over small-molecules. We open source all models introduced in this work as part of DeepChem, an open-source framework for deep-learning in drug discovery (Ramsundar, B. deepchem.io. https://github.com/deepchem/deepchem, 2016).