High-Throughput µPAD with Cascade Signal Amplification through Dual Enzymes for arsM in Paddy Soil.

The arsM gene is a critical biomarker for the potential risk of arsenic exposure in paddy soil. However, on-site screening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12a microfluidic paper-based analytical device (µPAD) was constructed for the high-throughput POU analysis of arsM, with cascade amplification driven by coupling crRNA-enhanced Cas12a and horseradish peroxidase (HRP)-modified probes. First, seven crRNAs were designed to recognize arsM, and their LODs and background signal intensities were evaluated. Next, a step-by-step iterative approach was utilized to develop and optimize coupling systems, which improved the sensitivity 32 times and eliminated background signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, and the assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reaction integration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying the pretreatment procedures and achieving high-throughput POU analysis. Finally, arsM in Wanshan paddy soil was screened on site, and the arsM abundance ranged from 1.05 × 106 to 6.49 × 107 copies/g; this result was not affected by the environmental indicators detected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and a microfluidic device was developed that contains many more channels than previous paper chips, greatly improving the analytical performance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales.

Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C-CN/PCNV heterojunction as emitter.

Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m6A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m6A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.

Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.

Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.

DNA digestion and formation of DNA-network structures with Holliday junction-resolving enzyme Hjc_15-6 in conjunction with polymerase reactions.

The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors.

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Method to generate Holliday junction recombination intermediates via RecA-mediated four-strand exchange.

DNA molecules that contain single Holliday junctions have served as model substrates to investigate the pathway in which homologous recombination intermediates are processed. However, the preparation of DNA containing Holliday junctions in high yield remains a challenge. In this work, we used a nicking endonuclease to generate gapped DNA, from which α-structured DNA or figure-8 DNA were created via RecA-mediated reactions. The resulting DNA molecules were found to serve as good substrates for Holliday junction resolvases. The simplified method negates the requirement for radioactive labelling of DNA, making the generation of Holliday junction DNA more accessible to non-experts.

CRISPR dual enzyme cleavage triggers DNA and RNA substrate cleavage for SARS-CoV-2 dual gene detection.

The widespread dissemination of coronavirus 2019 imposes a significant burden on society. Therefore, rapid detection facilitates the reduction of transmission risk. In this study, we proposed a multiplex diagnostic platform for the rapid, ultrasensitive, visual, and simultaneous detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 1ab (ORF1ab) and N genes. A visual diagnostic method was developed using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a/Cas13a dual-enzyme digestion system integrated with multiplex reverse transcriptase-recombinase polymerase amplification (RT-RPA). Two CRISPR-Cas proteins (Cas12a and Cas13a) were introduced into the system to recognize and cleave the N gene and ORF1ab gene, respectively. We used fluorescent or CRISPR double digestion test strips to detect the digested products, with the N gene corresponding to the FAM channel in the PCR instrument or the T1 line on the test strip, and the ORF1ab gene corresponding to the ROX channel in the PCR instrument or the T2 line on the test strip. The analysis can be completed in less than 20 min. Meanwhile, we assessed the application of the platform and determined a sensitivity of up to 200 copies/mL. Additionally, dual gene validation in 105 clinical nasopharyngeal swab samples showed a 100% positive predictive value agreement and a 95.7% negative predictive value agreement between our method and quantitative reverse transcription-polymerase chain reaction. Overall, our method offered a novel insight into the rapid diagnosis of SARS-CoV-2.

Identification of a leucine-zipper motif in pUL51 essential for HCMV replication and potential target for antiviral development.

Human cytomegalovirus (HCMV) can cause serious diseases in immunocompromised patients. Use of current antivirals is limited by their adverse effects and emergence of drug resistance mutations. Thus, new drugs are an urgent need. The terminase complex (pUL56-pUL89-pUL51) represents a target of choice for new antivirals development. pUL51 was shown to be crucial for the cleavage of concatemeric HCMV DNA and viral replication. Its C-terminal part plays a critical role for the terminase complex assembly. However, no interaction domain is clearly identified. Sequence comparison of herpesvirus homologs and protein modelling were performed on pUL51. Importance of a putative interaction domain is validated by the generation of recombinant viruses with specific alanine substitutions of amino acids implicated in the domain. We identified a Leucine-Zipper (LZ) domain involving the leucine residues L126-X6-L133-X6-L140-X6-L147 in C-terminal part of pUL51. These leucines are crucial for viral replication, suggesting the significance for pUL51 structure and function. A mimetic-peptide approach has been used and tested in antiviral assays to validate the interaction domain as a new therapeutic target. Cytotoxicity was evaluated by LDH release measurement. The peptide TAT-HK29, homologous to the pUL51-LZ domain, inhibits HCMV replication by 27% ± 9% at 1.25 µM concentration without cytotoxicity. Our results highlight the importance of a leucine zipper domain in the C-terminal part of pUL51 involving leucines L126, L133, L140 and L147. We also confirm the potential of mimetic peptides to inhibit HCMV replication and the importance to target interaction domains to develop antiviral agents.

Identification of small-molecule inhibitors of human MUS81-EME1/2 by FRET-based high-throughput screening.

The MUS81-EME1/2 structure-specific endonucleases play a crucial role in the processing of stalled replication forks and recombination intermediates, and have been recognized as an attractive drug target to potentiate the anti-cancer efficacy of DNA-damaging agents. Currently, no bioactive small-molecule inhibitors of MUS81 are available. Here, we performed a high-throughput small-molecule inhibitors screening, using the FRET-based DNA cleavage assay. From 7920 compounds, we identified dyngo-4a as a potent inhibitor of MUS81 complexes. Dyngo-4a effectively inhibits the endonuclease activities of both MUS81-EME1 and MUS81-EME2 complexes, with IC50 values of 0.57 µM and 2.90 µM, respectively. Surface plasmon resonance (SPR) and electrophoretic mobility shift assay (EMSA) assays reveal that dyngo-4a directly binds to MUS81 complexes (KD âˆ¼ 0.61 µM) and prevents them from binding to DNA substrates. In HeLa cells, dyngo-4a significantly suppresses bleomycin-triggered H2AX serine 139 phosphorylation (γH2AX). Together, our results demonstrate that dyngo-4a is a potent MUS81 inhibitor, which could be further developed as a potentially valuable chemical tool to explore more physiological roles of MUS81 in the cells.

Csx28 is a membrane pore that enhances CRISPR-Cas13b-dependent antiphage defense.

Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo-electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28's antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.

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.

Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system.

Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.

Terminase Subunits from the Pseudomonas-Phage E217.

Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas-phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ∼58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed an RNase H-like fold with two magnesium ions in the nuclease active site. Mutations in TerL residues involved in magnesium coordination had a dominant-negative effect on phage growth. However, the two ions identified in the active site were too far from each other to promote two-metal-ion catalysis, suggesting a conformational change is required for nuclease activity. We also determined a 3.38 Å cryo-EM reconstruction of E217 TerS that revealed a ring-like decamer, departing from the most common nonameric quaternary structure observed thus far. E217 TerS contains both N-terminal helix-turn-helix motifs enriched in basic residues and a central channel lined with basic residues large enough to accommodate double-stranded DNA. Overexpression of TerS caused a more than a 4-fold reduction of E217 burst size, suggesting a catalytic amount of the protein is required for packaging. Together, these data expand the molecular repertoire of viral terminase subunits to Pseudomonas-phages used for phage therapy.

8-Hydroxy-1,6-naphthyridine-7-carboxamides as Inhibitors of Human Cytomegalovirus pUL89 Endonuclease.

Human cytomegalovirus (HCMV) replication requires a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C) for viral genome packaging and cleavage. We have previously shown that pUL89-C can be pharmacologically inhibited with designed metal-chelating compounds. We report herein the synthesis of a few 8-hydroxy-1,6-naphthyridine subtypes, including 5-chloro (subtype 15), 5-aryl (subtype 16), and 5-amino (subtype 17) variants. Analogs were studied for the inhibition of pUL89-C in a biochemical endonuclease assay, a biophysical thermal shift assay (TSA), in silico molecular docking, and for the antiviral potential against HCMV in cell-based assays. These studies identified eight analogs of 8-hydroxy-1,6-naphthyridine-7-carboxamide subtypes for further characterization, most of which inhibited pUL89-C with single-digit µM IC50 values, and conferred antiviral activity in µM range. TSA and molecular modeling of selected analogs corroborate their binding to pUL89-C. Collectively, our biochemical, antiviral, biophysical and in silico data suggest that 8-hydroxy-1,6-naphthyridine-7-carboxamide subtypes can be used for designing inhibitors of HCMV pUL89-C.

A protocol to determine the activities of human MUS81-EME1&2 endonucleases.

The human MUS81-EME1&2 complexes are structure-selective endonucleases that play important roles in DNA damage repair. Here, we describe a protocol to determine the endonuclease activities of MUS81-EME1&2 complexes toward various DNA structures. We co-express MUS81 with EME1 or EME2 and purify the complexes with high purity, and determine their activities on the cleavages of 3' flaps, 5' flaps, nicked double-stranded DNAs, and Holliday junctions. This protocol can also be used for the determination of substrate preferences of other structure-selective endonucleases. For complete details on the use and execution of this protocol, please refer to Hua et al. (2022).

Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2.

Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/µL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

Discovery of deoxyribonuclease II-like proteins in bacteria.

Deoxyribonuclease II (DNase II) is one of the earliest enzymes discovered in the history of biochemistry. Its role in apoptosis and development has been documented with great detail in eukaryotes. Prior in silico analyses showed its complete absence in bacterial genomes, with the exception of single bacterial genus: Burkholderia. It is therefore considered to be a eukaryotic enzyme. Here we show that the presence of DNase II is not limited to Burkholderia, as we find over one hundred DNase II-like sequences spanning 90 bacteria species belonging to 54 different genera and seven phyla. The majority of the significant hits (85%) come from Bacteroidetes and Proteobacteria phyla. Sequence analyses reveal that bacterial DNase II-like proteins possess a signature catalytic motif of eukaryotic DNase II. In phylogenetic analyses, we find that bacterial DNase II-like proteins are divided into two distinct clades. Our structural analyses reveal high levels of similarity between experimentally determined crystal structures of recombinant Burkholderia thailandensis DNase II and candidate bacterial DNase II-like proteins. We also biochemically show that Chromobacterium violaceum cell lysate possesses acidic DNase II-like activities. Collectively, our results indicate that DNase II has deeper evolutionary roots than previously thought. We argue that either some prokaryotic lineages have undergone losses of DNase II genes, resulting in rare conservation, or some lineages have acquired DNase II genes from eukaryotes through lateral gene transfer. We also discuss the possible involvement of DNase II as a part of an anti-phage defense system in bacteria.

Crystal structure of the human MUS81-EME2 complex.

MUS81 is an important structure-specific endonuclease responsible for the processing of stalled replication forks and recombination intermediates. In human, MUS81 functions by forming complexes with its regulatory subunits EME1 and EME2, playing distinct roles in G2/M and S phases. Although the structures of MUS81-EME1 have been intensively studied, there is no structure information available about MUS81-EME2. Here, we report the crystal structure of MUS81-EME2, which reveals an overall protein fold similar to that of MUS81-EME1 complex. Further biochemical and structural characterization shows that the MUS81-EME1 and MUS81-EME2 complexes are identical in substrate recognition and endonuclease activities in vitro, implying that the distinct cellular roles of the two complexes could arise from temporal controls in cells. Finally, an extensive structure-guided mutagenesis analysis provides implications for the molecular basis of how the MUS81-EME endonucleases recognize various DNA substrates in a structure-selective manner.

Assembly of Cas7 subunits of Leptospira on the mature crRNA of CRISPR-Cas I-B is modulated by divalent ions.

The spirochete Leptospira interrogans serovar Copenhageni harbors the genetic elements of the CRISPR-Cas type I-B system in its genome. CRISPR-Cas is a CRISPR RNA (crRNA) mediated adaptive immune system in most prokaryotes against mobile genetic elements (MGEs). To eliminate the intruding MGEs, CRISPR-Cas type I systems utilize a Cascade (CRISPR-associated complex for antiviral defense) complex composed of Cas5, Cas6, Cas7, and Cas8 bound with a crRNA. The Cas7 is essentially known to constitute the major component of the Cascade complex. The present study reports the biochemical characterization of the Cas7 (LinCas7) from the CRISPR-Cas type I-B system of L. interrogans serovar Copenhageni. The pure recombinant LinCas7 (rLinCas7) exists as a monomer in the solution by size exclusion chromatography. The rLinCas7 demonstrates an endoDNase activity dependent upon divalent Mg2+ ions, monovalent ions, pH, temperature, and substrate size. Analysis of ribonucleoprotein composite (rLinCas7-crRNA) by electron microscopy and native-PAGE demonstrated that rLinCas7 could oligomerize on the mature CRISPR RNA (crRNA) framework in the presence of Mg2+ ions. The ribonucleoprotein composite attains a helical shape similar to the backbone of the Cascade complex. However, in the absence of Mg2+ ions, rLinCas7 acts as an RNase. The fluorescence spectroscopy disclosed a weak interaction (Kd = 26.81 mM) between rLinCas7 and Mg2+ ions, leading to an overall conformational change in rLinCas7 that modulates the rLinCas7's activity on DNA and RNA substrates. The nuclease activity of LinCas7 characterized in this study aids to the functional divergences among proteins of the Cas7 family from different CRISPR-Cas systems in various organisms.