Structure and engineering of the minimal type VI CRISPR-Cas13bt3.

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.

Fanzor is a eukaryotic programmable RNA-guided endonuclease.

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 cleavage1. Although a few eukaryotic RNA-guided systems have been studied, including RNA interference2 and ribosomal RNA modification3, it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided systems (termed OMEGA) was reported4,5. The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity4,6. TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins4,7, 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.

Cryo-EM structure of the transposon-associated TnpB enzyme.

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. 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.

Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA.

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 technologies1,2. Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development3. 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 domain4. 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.

Diversity, evolution, and classification of the RNA-guided nucleases TnpB and Cas12.

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.

The Transposon-Encoded Protein TnpB Processes Its Own mRNA into ωRNA for Guided Nuclease Activity.

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.

Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering.

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.

Compact RNA editors with small Cas13 proteins.

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.

Structure of the IscB-ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9.

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.

The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases.

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 element­guided activity), with strong potential for developing as biotechnologies.

High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage.

Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells.

Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data.

Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process. In fact, some promoters that are often thought of as constitutive can show changes in activity when growth conditions change. For these reasons, we have developed a system of ordinary differential equations for estimating dynamic promoter activity for promoters that change their activity in response to the environment that is robust to noise and changes in growth rate. Our approach, inference of dynamic promoter activity (PromAct), improves on existing methods by more accurately inferring known promoter activity profiles. This method is also capable of estimating the correct scale of promoter activity and can be applied to quantitative data sets to estimate quantitative rates.

Glucose-Dependent Promoters for Dynamic Regulation of Metabolic Pathways.

For an industrial fermentation process, it can be advantageous to decouple cell growth from product formation. This decoupling would allow for the rapid accumulation of biomass without inhibition from product formation, after which the fermentation can be switched to a mode where cells would grow minimally and primarily act as catalysts to convert substrate into desired product. The switch in fermentation mode should preferably be accomplished without the addition of expensive inducers. A common cell factory Saccharomyces cerevisiae is a Crabtree-positive yeast and is typically fermented at industrial scale under glucose-limited conditions to avoid the formation of ethanol. In this work, we aimed to identify and characterize promoters that depend on glucose concentration for use as dynamic control elements. Through analysis of mRNA data of S. cerevisiae grown in chemostats under glucose excess or limitation, we identified 34 candidate promoters that strongly responded to glucose presence or absence. These promoters were characterized in small-scale batch and fed-batch cultivations using a quickly maturing rapidly degrading green fluorescent protein yEGFP3-Cln2PEST as a reporter. Expressing 3-hydroxypropionic acid (3HP) pathway from a set of selected regulated promoters allowed for suppression of 3HP production during glucose-excess phase of a batch cultivation with subsequent activation in glucose-limiting conditions. Regulating the 3HP pathway by the ICL1 promoter resulted in 70% improvement of 3HP titer in comparison to PGK1 promoter.