Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex generates two cyclic oligoadenylates  that allosterically activate ancillary nucleases. We show that both Can1 and Can2 nucleases cleave single-stranded RNA, single-stranded DNA, and double-stranded DNA in the presence of cA4. We integrate the Can2 nuclease with type III-A RNA capture and concentration for direct detection of SARS-CoV-2 RNA in nasopharyngeal swabs with 15 fM sensitivity. Collectively, this work demonstrates how type-III CRISPR-based RNA capture and concentration simultaneously increases sensitivity, limits time to result, lowers cost of the assay, eliminates solvents used for RNA extraction, and reduces sample handling.Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we repurpose the type III-A CRISPR complex from  Many viral diagnostic approaches require nucleic acid extraction and amplification prior to detection. Here, the authors develop a method based on type III CRISPR systems which allows sequence specific capture, concentration, and detection of viral RNA directly from patient samples. While the complexity and turnaround times necessary for qPCR are acceptable for many diagnostic applications, the SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) pandemic reveals an urgent need for diagnostics that are easy to distribute, simple to perform, and fast enough to stop transmission of a contagious disease1. Although rapid antigen tests and isothermal amplification methods have helped address this need, these and other emerging methods have limitations related to sensitivity, versatility, or specificity3.Although qPCR (quantitative polymerase chain reaction) remains the “gold standard” for nucleic acid detection, it requires sophisticated equipment, trained personnel, efficient specimen transport to high-complexity labs, and reliable reporting systems5. The first CRISPR-based viral diagnostic came from Collins and colleagues in 2016, when they demonstrated that Cas9 could be used to discriminate between different variants of the Zika virus6. This approach relies on converting viral RNA to DNA using reverse transcriptase, followed by isothermal DNA amplification prior to sequence-based discrimination by Cas9. The exclusive recognition of double-stranded DNA (dsDNA) by Cas9 seemed to be an intrinsic limitation for diagnostic applications that require RNA detection. However, Beisel and colleagues recently developed a creative method that uses the trans-acting CRISPR-RNA (tracrRNA) to capture complementary RNA guides derived from RNA viruses7. In this system, the engineered tracrRNA-crRNA hybrid guides Cas9 to a complementary dsDNA reporter. While this approach enables RNA detection, Cas9 is a single turn-over enzyme, which may limit its sensitivity. In contrast to Cas9, target recognition by type V (Cas12-DETECTR) and type VI (Cas13-SHERLOCK) CRISPR-systems activates a multi-turnover non-sequence-specific “collateral nuclease” activity that amplifies the signal by cleaving thousands of reporter molecules for every target bound9. The recently reported rates of collateral cleavage by target bound Cas12 and Cas13 nucleases indicate that the theoretical limit of detection, without prior amplification of the target is 1 pM (~106 copies/µL)11.CRISPR RNA-guided diagnostics (CRISPR-dx) are a diverse group of nascent technologies that aim to address current limitations by providing a versatile and programmable platform that is sufficiently sensitive for clinical applications and stable enough for distribution14. The polymerase domain has been estimated to generate ~1000 cyclic oligoadenylates per bound RNA15, which trans-activate multi-turnover ancillary nucleases that provide defense from invading genetic parasites17. Thus, this biochemical cascade provides two signal amplifications steps, the second of which activates thousands of multi-turnover ancillary nucleases, rather than relying on a single collateral nuclease  bound to a target. However, initial efforts to implement this approach failed to be sufficiently sensitive for clinical applications without prior amplification of the target RNA20. The sensitivity of this first-generation diagnostic was in part limited by the use of an ancillary nucleases  that also degrade the cyclic nucleotide activator25. Recently, Malcolm White’s lab demonstrated that alternative ancillary nucleases, which efficiently cleave reporters but do not cleave the signaling molecule, can be used to enhance the sensitivity of type III-based diagnostics26.Like type VI, type III CRISPR systems also recognize RNA. However, unlike any other system, target recognition by type III complexes simultaneously activates polymerase and HD-nuclease domains in the Cas10 subunit4). We demonstrate how the type III complex can be used to bypass RNA extraction methods, and that coupling type III-based RNA capture with the AaCan2 nuclease further increases the sensitivity of SARS-CoV-2 RNA detection in patient swabs to 15 fM (8.3 × 103 copies/µL).Despite innovations leading to new and improved CRISPR-based diagnostics, point-of-care testing requires new strategies that simplify the workflow and increase the sensitivity without prior RNA purification or amplification . Here, we bring CRISPR-dx closer to a deployable diagnostic by developing a type III CRISPR-based method for sequence-specific capture and concentration of a specific RNA from a complex mixture. To improve the sensitivity, we purify several different ancillary nucleases , systemically test nuclease activation using a series of purified cyclic oligoadenylate standards, and test for ring nuclease activity. We show that Can1 and Can2 nucleases cleave single-stranded RNA (ssRNA) and DNA (ssDNA), as well as double-stranded DNA (dsDNA) in the presence of a Cas10-generated cyclic oligomer composed of four adenosines  that form the oligomeric “backbone” of the complex28. Type III complexes release fragments of the cleaved target, which inactivates ATP polymerization by the Cas10 subunit28. Previously, we mutated residues in the Csm3 subunit responsible for target RNA cleavage (D34A), purified the RNase-dead complex (TtCsmCsm3-D34A), and showed that the mutant complex provides more sensitive detection of viral RNA than the wild-type complex18. To further increase the sensitivity, we set out to determine if TtCsmCsm3-D34A could be used to concentrate sequence-specific RNAs. To test this approach, we mixed 32P-labeled target or non-target RNAs with TtCsmCsm3-D34A, incubated for 20 min, and concentrated the His-tagged complex using nickel-derivatized magnetic beads . Type III CRISPR-based concentration increases the amount of cA3 and cA4, as compared to reaction performed without RNA concentration  bind and cleave complementary single-stranded RNA (ssRNA) targetsads Fig. . The beaads Fig. . To deteion Fig. .Fig. 1Ty4-activated ribonuclease, to generate a real-time fluorescent readout for Csm-based RNA detection18 . Csm-based RNA enrichment increased the sensitivity of the assay 100-fold compared to the assay without the pull-down  and a carboxy-terminal HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domain33. Like Csm6 proteins, Can proteins also contain amino-terminal CARF domains, but the carboxy-terminal nucleases are distinct. The Can1 protein from Thermus thermophilus (TtCan1) has a unique monomeric architecture with two non-identical CARF domains, one nuclease-like domain (NLD), and one restriction endonuclease domain (PD-(D/E)XK)33, while Can2 nucleases contain a single CARF domain and form symmetrical homodimers32  are another family of recently identified proteins that are activated by cyclic oligoadenylates and lack ring nuclease activitys32 Fig. .Fig. 2CaThermus thermophilus . Elevated temperatures are anticipated to improve accessibility to RNA targets that might otherwise be obscured by secondary structures and the use of thermostable ancillary nucleases may enable detection of a specific RNA in a single tube at a single temperature. In addition, the stability of these proteins34, may have downstream benefits when it comes to packaging and distribution.To identify Can1 and Can2 orthologs, we generated hidden Markov models (HMMs) to query publicly available microbial genomes and metagenomes from NCBI and JGI. This analysis identified 204 Can1 and 3121 Can2 proteins. Based on this analysis, we selected one Can1 protein and three Can2 proteins from thermophilic organisms. We hypothesized that thermostable nucleases will be compatible with RNA detection and target RNA-activated polymerization of ATP by the Csm complex from 33, TtCan1 exhibits Mn2+-dependent linearization of supercoiled plasmid DNA in the presence of cA4  might allosterically activate the nuclease activity. We tested a library of twelve chemically synthesized cyclic oligonucleotides in a reaction with TtCan1 and three different substrates  , Thermus thermophilus (TtCan2), and Archaeoglobi archaeon JdFR-42 (AaCan2) were cloned and expressed in E. coli   drop in the activity. This distinction in activity between the enzymes is consistent with the ring-nuclease activity of TtCsm6 rapidly degrading its activator, while AaCan2 binds and preserves the cyclic nucleotide  Fig. . Moreove4, which stably activates AaCan2, whereas the ring-nuclease of TtCsm6 rapidly degrades cA4 limiting the background signal. To test this hypothesis, we titrated concentration of TtCsmCsm3-D34A-complex and show that 50-fold less of the complex  reduces the background without compromising target-specific activity, which significantly improves signal-to-noise   Fig. . Collect4-activated collateral cleavage of ssRNA reporters, the TtCsmCsm3-D34A complex also produces cA3  has the highest dsDNase activity and digests plasmid DNA into 300–400 bp fragments in the presence of cA3  could be used to capture target RNA directly from patient samples, without prior RNA extraction. Sequence-specific capture and concentration directly from patient samples could drastically reduce time, reagents, and equipment costs.Most CRISPR-based diagnostics assays reported to date use purified RNA as the starting material for viral detection assaysTo address this challenge, we set out to incorporate “capture and concentration” into the fluorescent-based detection method. However, the magnetic beads used to pull-down target RNA unexpectedly obscure the fluorescent signal released when AaCan2 cleaves the reporter , and chelating agents , to identify lysis conditions compatible with the two-step detection assay that relies on Csm-based RNA capture, followed by detection with AaCan2. The lysis buffers tested do not inhibit the assays with purified RNA and increase the signal-to-noise ratio   Fig. .5 copies/µL) . No cross-reactivity is detectable with RNAs from Influenza B, Human respiratory syncytial virus (RSV), and HCoV-229E .Finally, to test cross-reactivity of TtCsm-based RNA capture coupled with AaCan2-based fluorescent detection, we use a panel of seven respiratory viruses, including SARS-CoV-1, Middle East respiratory syndrome coronavirus (MERS-CoV), seasonal coronaviruses 229E (HCoV-229E), NL63 (HCoV-NL63), and HKU1 (HCoV-HKU1). To perform the assay, we used the highest concentration of genomic RNAs available at ATCC and recommended by FDA  and type VI (Cas13) CRISPR systems, and the sensitivity of these techniques has improved from picomolarWhile sensitivity continues to improve, less attention and little progress has been made to develop methods that bypass RNA extraction. Purifying nucleic acids from patient samples prior to testing requires specialized equipment that increases the cost, labor, and time-to-result. Thus, testing protocols that require RNA extraction represent a major limitation for point-of-care implementation, which is critical for limiting the spread of a contagious agent. Here, we demonstrate how type III CRISPR systems can be used for sequence-specific capture and detection of viral RNA directly from unprocessed patient samples without pre-amplification.19. More recent improvements using different type III complexes or different ancillary nucleases have been used to detect spiked SARS-CoV-2 RNA with ~2–4 fM sensitivity in 30 min26  in clinical samples without laboratory-based RNA extraction or pre-amplification in less than 20 min total. While the sensitivity of the approach is still inferior to RT-qPCR, it is sufficient to identify infected individuals capable of spreading SARS-CoV-239 and is comparable to rapid antigen tests2.In 2021, the first attempts to repurpose type III CRISPR systems for SARS-CoV-2 diagnostics achieved 0.1–1 nM sensitivity of RNA detection without pre-amplification4. While methods that incorporate tandem CRISPR nucleases  are currently more sensitive (~50 aM), the intrinsic amplification of RNA recognition by type III systems may ultimately improve sensitivity. Type III systems uniquely amplify RNA recognition in two sequential steps: first, through Cas10-mediated polymerization of cOAs, and second, through cOA-mediated activation of multi-turnover effectors . In addition to the advantages that might come from consecutive stages of signal amplification, the separation of target recognition by the type III surveillance complex  from collateral cleavage by ancillary effectors also enables programmable RNA capture. Unlike Cas13, which relies on the same active site for target and non-target collateral cleavage40, the RNase-dead TtCsm complex (TtCsmCsm3-D34A) can capture and maintain target RNA from a larger volume and concentrate these RNAs for various downstream applications. Incorporating RNA capture increases the sensitivity of type III CRISPR-based diagnostic and allows direct detection in clinical samples without RNA extraction, a prerequisite for most current platforms. Further, the separation of on-target (cis-) and collateral (trans-) activities in type III CRISPR systems provides a flexible platform for creating combinatorial assemblies of detection  and readout  modules based on diverse CARF-, SAVED- (SMODS-associated and fused to various effector domains)16 or CBASS  proteins for applications in diagnostics and beyond.Like Cas13, type III systems also recognize RNA, and the most sensitive detection methods developed to date for either approach rely on collateral nuclease activity to release a fluorescent signal5 copies/µL) we detect a weak, but reproducible signal to some of the coronaviruses  but not to HCoV-229E or other respiratory viruses . Each of the coronaviruses have 4–7 mismatches in first two segments (S1–S2) of crRNA:target duplex , while HCoV-229E, which generates no signal, has an additional mismatch at the 1st position. The first two nucleotides in segment S1 have the greatest effect on cOA production41, therefore the additional mismatch in HCoV-229E at the 1st position might explain the complete loss of signal, as compared to the weak signal generated by the other coronaviruses, which contain two mismatches in segment 1 (S1). Our original bioinformatic pipeline for crRNA design filtered out guides with potential cross-reactivity, however, we prioritized the total number of mismatches in segments S1 and S2 rather than the position of these mismatches18. The cross-reactivity assay in this manuscript and published biochemical data41 demonstrate the importance of mismatch positions +1 and +2, which is expected to improve the specificity of the next generation of guides. Overall, we anticipate that type III-based RNA pull-down techniques that bypass RNA extraction, combined with further optimization of lysis conditions, more efficient guide design, and the integration of next generation of signal detection methods  will help to bring type III CRISPR diagnostics closer to current standards of rapid molecular testing.Direct detection of RNA in clinical samples without RNA extraction or pre-amplification will advance direct-to-consumer diagnostics. However, eliminating pre-amplification removes additional selectivity imparted by the traditional use of primers. To evaluate cross-reactivity of TtCsm-based RNA capture coupled with AaCan2-based fluorescent detection, we tested a panel of seven respiratory viruses. At high RNA concentrations  For the Protection of Human Subjects (FWA 00000165). De-identified clinical samples were obtained with IRB approval (protocol #DB033020) and informed consent from patients undergoing testing for SARS-CoV-2 at Bozeman Health Deaconess Hospital.Nasopharyngeal swabs from patients that either tested negative or positive for SARS-CoV-2 were collected in viral transport media. RNA was extracted from all patient samples using the QIAamp Viral RNA Mini Kit (QIAGEN).2-6), sodium salts of cyclic tri- and tetra- guanosine monophosphates , sodium salts of cyclic (guanosine- (2′ −> 5′)- monophosphate- adenosine- (3′ −> 5′)- monophosphate) (cGAMP), cyclic adenosine monophosphate-guanosine monophosphate (cAG), and cyclic diguanosine-5′-monophosphate (cG2) were purchased from Biolog Life Science Institute. Fluorescent reporters (RNA and DNA) were purchased from IDT  were used as negative controls. RNA extracted from HEK 293T cells was diluted to match the average Ct level (~27) obtained for RNAseP mRNA in RNA samples extracted from nasopharyngeal swabs , were a gift from Jennifer Doudna. Vector pCDF-5xT7-TtCsm was used as a template for site-directed mutagenesis to mutate the D34 residue in Csm3 to alanine (D34A) and inactivate Csm3-mediated cleavage of target RNA (pCDF-5xT7-TtCsmCsm3-D34A)42. The CRISPR array in pACYC-TtCas6-4xcrRNA4.5 was replaced with a synthetic CRISPR array (GeneArt) containing five repeats and four identical spacers, designed to target the N-gene of SARS-CoV-2 18. TtCas6 was PCR was PCR-amplified from the pACYC-TtCas6-4xcrRNA4.5 plasmid and cloned between the NcoI and XhoI sites in the pRSF-1b backbone (Millipore Sigma) (pRSF-TtCas6). Expression vector encoding TtCsm6 nuclease, pC0075 TtCsm6 His6-TwinStrep-SUMO-BsaI, was a gift from Feng Zhang (Addgene plasmid #115270)43.Plasmids encoding the type III-A Csm complex from Thermus thermophilus , Can2 from Archaeoglobi archaeon JdFR-42 , Clostridium thermobutyricum , and Thermus thermophilus , were codon optimized for expression in E. coli, synthesized by GenScript, and cloned into pC0075 vector (Addgene #115270) in frame with the N-terminal His6-TwinStrep-SUMO tag using NcoI and XhoI restriction sites to replace the TtCsm6 gene. NucC from Clostridium tepidum BSD2780120874b_170522_A10 , Elioraea sp. Yellowstone  and Acidimicrobiales bacterium mtb01 , were cloned into pC0075 backbone using the same restriction sites as for Can1 and Can2 genes.Gene fragments encoding for Can1 from Csm3-D34A complex and TtCsm6 were performed as previously described18. TtCan1, AaCan2, CtCan2, TtCan2, CtNucC, EsNucC, and Amtb01NucC) were purified according to the following protocol. Each expression vector was transformed into Escherichia coli BL21(DE3) cells and grown in LB Broth (Lennox) (Thermo Fisher Scientific) at 37 °C to an OD600 of 0.5. Cultures were then incubated on ice for 1 h, and then induced with 0.5 mM IPTG for overnight expression at 16 °C. Cells were lysed with sonication in Lysis buffer  and lysate was clarified by centrifugation at 10,000 × g for 25 min, 4 °C. The lysate was heat-treated at 55 °C for 45 min and clarified by centrifugation at 10,000 × g for 25 mins at 4 °C. His6-TwinStrep-tagged protein was bound to a StrepTrap HP column (Cytiva) and washed with Lysis buffer. The protein was eluted with Lysis buffer supplemented with 2.5 mM desthiobiotin and concentrated (10k MWCO Corning Spin-X concentrators) at 4 °C. Affinity tags were removed from the protein using His-tagged SUMO protease (100 µL of 2.5 mg/mL protease per 20 mg of protein) during dialysis against SUMO digest buffer , 0.15% Igepal) at 4 °C overnight. The tag and the protease were applied to HisTrap HP column (Cytiva), and the flow-through was concentrated using Corning Spin-X concentrators at 4 °C. Finally, the protein was purified using a HiLoad Superdex 200 26/600 size-exclusion column (Cytiva) in storage buffer . Fractions containing the target protein were pooled, concentrated, aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C.Expression and purification of the TtCsm32P]-ATP (PerkinElmer) for 30 min at 37 °C. The kinase was heat inactivated by heating at 65 °C for 20 min.Target SARS-CoV-2 N1) and non-target RNAs were transcribed from PCR extended duplex oligos using home-made T7 RNA polymerase (Supplementary Table  N1 and n32P-labeled RNA (25 nM) was incubated with TtCsmCsm3-D34A (160 nM) targeting SARS-CoV-2 N-gene in 1× Binding Buffer  for 20 min at 65 °C. The reaction mixtures were added to 10 µL of HisPur Ni-NTA Magnetic beads (ThermoFisher) equilibrated in Binding Buffer and incubated on ice 30 min with vortexing every 10 min. The beads were separated from the supernatant using a magnet and washed with 50 µL 1× binding buffer. The RNA was extracted from supernatant (unbound fraction) and beads (bound fraction) using Acid Phenol: chloroform (Ambion). Extracted RNA was resolved using UREA-PAGE, exposed to a phosphor screen, and imaged on a Typhoon 5 imager (Amersham). Bands corresponding to the IVT RNAs were quantified using ImageJ v1.52t and the percent bound calculated [bound/(bound + free)*100%].For the experiments shown in Fig. D34A complex (25 nM) in 1× Binding Buffer (V = 50 µL) and incubated for 30 min on ice. The beads with the complex (Csm-beads) were concentrated with a magnet and resuspended in 5 µL of 1× Binding Buffer.The HisPur Ni-NTA Magnetic beads (ThermoFisher) were washed two times with a 1× Binding Buffer . For one reaction, 5 µL of equilibrated beads were mixed with TtCsm10 copies/µL) or 3 µL of NTC were mixed with TtCsmCsm3-D34A complex (25 nM) and 250 µM ATP supplemented with [α-32P]-ATP (PerkinElmer) in the reaction buffer phosphine)), 5 mM magnesium sulfate). The reaction was incubated at 60 °C for 1 h. For the pull-down reactions, 120 µL of positive or negative samples were mixed with 5 µL of Csm-beads in Binding Buffer  for 10 min at 60 °C. The Csm-beads were concentrated with a magnet and the supernatant was discarded. The Csm pellets were resuspended in 30 µL of the reaction buffer and 250 µM ATP supplemented with [α-32P]-ATP (PerkinElmer). Reaction products were phenol-chloroform extracted and resolved on silica TLC plates (Millipore).For the experiments shown in Fig. Samples (1 µL) were mixed with 100 mM sodium acetate, pH 5.2 (2 µL), and spotted 1.5 cm above the bottom of the TLC plate. The plate was placed inside a 2 L beaker filled to ~0.5 cm with developing solvent  and capped with aluminum foil. The plate was run for 2 h at room temperature and dried. TLC plate was exposed to a phosphor screen and imaged with Typhoon phosphor imager. Chemically synthesized standards (2 µM) were resolved on the same TLC plate and visualized using UV shadowing.3 and cA4 hydrolysis in the presence of ancillary nuclease, radiolabeled cA3 and cA4 produced above were mixed with nuclease (500 nM) in the reaction buffer and incubated for 1 hour at 55 °C. Reaction products were phenol-chloroform extracted and resolved using TLC for 45 min as described above.To test cAD34A complex, 300 nM of nuclease  with corresponding reporter in a reaction buffer phosphine)), 5 mM magnesium sulfate (for TtCsm6 and CtNucC) or 5 mM manganese(II) chloride (for AaCan2) in a 30 µL reaction. The reporter B8 (300 nM) was used for the reaction with TtCsm6, D7 (300 nM) – with AaCan2, and dsDNA probe (300 nM) – with CtNucC. Reactions were incubated at 55 °C or 60 °C. Cleavage of fluorescent reporters was detected by measuring fluorescence every 10 sec in a real-time PCR instrument QuantStudio 3 (Applied Biosystems).3 µL of RNA sample was mixed with 250 µM ATP, 0.5 nM TtCsmD34A complex with the magnetic beads, the HisPur Ni-NTA Magnetic beads (ThermoFisher) were washed two times with a 1× Binding Buffer . 5 µL of equilibrated beads were mixed with TtCsmD34A complex (30 nM) in 1× Binding Buffer (V = 50 µL) and incubated for 30 min on ice. The beads with the complex (Csm-beads) were concentrated with a magnet and resuspended in 5–500 µL of 1× Binding Buffer. For one reaction 5 µL of resuspended Csm-beads was used.To bind TtCsm120 µL of sample was mixed with 5 µL of Csm-beads in 1× Binding Buffer for 10 min at 60 °C . In brief, 20 µL reaction included 8.5 µL of Nuclease-free Water, 1.5 µL of Primer and Probe mix , 5 µL of TaqPath 1-Step RT-qPCR Master Mix  and 5 µL of the RNA. Nuclease-free water was used as negative template control (NTC). Amplification was performed as follows: 25 °C for 2 min, 50 °C for 15 min, 95 °C for 2 min followed by 45 cycles of 95 °C for 3 s and 55 °C for 30 s. To quantify viral RNA in the samples, standard curve for N1 primers was generated using a dilution series of a SARS-CoV-2 synthetic RNA fragment  spanning N gene with concentrations ranging from 10 to 106 copies per µL. Three technical replicates were performed at each dilution. The NTC showed no amplification throughout the 45 cycles of qPCR.RT-qPCR was performed using N1 and RP CDC primers . RNA was extracted from patient samples with QIAamp Viral RNA Mini Kit  and used for one-step RT-qPCR in ABI 7500 Fast Real-Time PCR System according to CDC protocols (DNA cleavage fragments were sequenced using Oxford Nanopore with Ligation Sequencing Kit (SQK-LSK109). After incubation with nucleases, cleavage fragments were column-purified using DNA Clean & Concentrator-5 kit  as instructed. Next, for each sample 200 ng of purified DNA was used to prepare sequencing libraries with NEBNext® Ultra™ II DNA Library Prep Kit . Briefly, DNA was end-repaired with NEBNext Ultra II End Prep Enzyme Mix, which fills 5′- and removes 3′- overhangs. Next, end-repaired fragments were barcoded with Native Barcoding Expansion kit  using Ultra II Ligation Master Mix (NEB). Barcoded DNA fragments were pooled together and purified with magnetic beads . Freshly mixed 80% ethanol was used to wash magnetic bead pellet. Sequencing adapters (AMII) were ligated to barcoded DNA using NEBNext® Quick Ligation Module . Ligation reactions were purified with magnetic beads. SFB buffer  was used for washes. Resulting DNA library was eluted from the beads in 20 µL of EB buffer . DNA concentration was measured with Qubit dsDNA HS Assay , and 20 ng was loaded on the Nanopore MinION . The flow cell was primed, and library was loaded according to Oxford Nanopore protocol (SQK-LSK109 kit). The sequencing run was performed in the fast base calling mode in the MinKNOW software v5.2.4.bamtobed function in bedtools package v2.30.0 was used to generate BED files and read coordinates were extracted. Read end coordinates were used to calculate cleavage fragment length distributions and map frequencies of cuts at specific locations. To analyze the sequence preferences of each nuclease, 14 bp windows surrounding read ends were extracted with getfasta function from bedtools package v2.30.0. Resulting fasta files were used to calculate position weigh matrices (PWMs) with getPwmFromFastaFile function in DiffLogo v2.16.0R package. Finally, PWMs were plotted as sequence logos using ggseqlogo v0.1R package. Sequencing depth around the most frequent cut site for each nuclease was calculated with samtools v1.13 depth function and plotted with ggplot2 package v3.3.5 in RStudio v2022.07.1+554.Sequenced reads were demultiplexed using guppy-barcoder v6.2.11+e17754edc (ONT) and aligned with minimap2 v2.17-r954-dirty (-ax map-ont mode) to the reference plasmid sequence that was modified by adding 1000 bp overlaps at the 5′- and 3′- ends. Overlapping regions were introduced to account for circular nature of the plasmid. Resulting alignments (BAM files) were sorted and indexed using samtools v1.13. Next, To determine the optimal RNA or DNA reporter for each cOA-activated nuclease, we constructed a library of variable single-stranded RNA or DNA molecules tethering a FAM fluorophore to an Iowa Black quencher. The Biostrings package in R was used to construct a library of reporter sequences containing each of the 64 unique trinucleotide combinations possible. Since multiple unique trinucleotides could be included in a single reporter , we narrowed our initial library of 64 reporters to remove redundant sequences. This resulted in a library of 24 unique reporter sequences, each of which were integrated into reporters of different length  plasmid with TtCan1, AaCan2 or CtNucC (15–200 nM) in the presence of cOAx (20–50 nM) in 10 μL reaction. After 5–15 min incubation, Gel Loading Dye, Purple (6X) (NEB) was added and 4 µL was loaded on 1% agarose gel. For ssDNA and ssRNA cleavage assays, 0.425 µM of 71 nt DNA oligo , 0.425 µM of 74 nt IVT RNA SARS-CoV-2 N-gene . The generated alignments for Can1 and Can2 were curated with MaxAlign v1.146 to remove misaligned or non-homologous sequences. The resulting dataset—comprised of 29 Can1-like and 1,283 Can2-like proteins, respectively—were then individually realigned with MAFFT and HMMbuild47 (HMMER v3.2.1) was used to generate HMM profiles from each alignment. The resulting profiles were used to search a local database of prokaryotic genomes from NCBI  and list of sequences identified in BLAST search from previous steps. An initial search performed with these HMM profiles identified 1442 Can1 and 5,431 Can2 homologs, which were manually filtered according to the presence of domains that define each protein, as well as the presence of conserved residues found in CARF and nuclease domains. The resulting set of 204 Can1 and 3,121 Can2 proteins were merged into a single file and aligned in MAFFT (LINSI option) for downstream phylogenetic analyses. Next, Trimal v1.448 was used to remove columns in the alignment comprised of ≥70% gaps. Thermostable homologs of Can1 and Can2 were annotated according to organisms that they are originated. ProtTest v3.4.249 was used to select an evolutionary model, and a phylogenetic tree was constructed in IQ-TREE v1.6.150 using the recommended model . The phylogenetic tree was plotted using the ggTree package in R51.A DELTA-BLAST was initiated, using previously described Can1 and Can2 proteins as queries35 generated a list of closely related proteins . The resulting dataset was filtered with CD-HIT v4.7 to remove redundant sequences. The resulting 1230 NucC sequences were aligned with MAFFT , and poorly aligned and highly gapped sequences were removed with MaxAlign. The resulting set of 896 NucC sequences were re-aligned with MAFFT as previously described, and the resulting alignment was used to generate a NucC HMM profile which we used to search within prokaryotic genomes from NCBI. This search identified 1774 hits, which were filtered according to the presence of restriction endonuclease-like domain , gate-loop and cA3 binding domains and were aligned with MAFFT. The remaining NucC homologs were curated according to organisms they are originated from to identify thermostable NucC homologs. The resulting alignment of 1510 NucC proteins with 21 thermostable homologs was used to generate a phylogenetic tree with FastTree v2.1.1052 and was plotted using the ggTree package in R.A phylogenetic tree of NucC proteins was generated using the same methods as described above for Can1/Can2 proteins. Briefly, DELTA-BLAST and PSI-BLAST searches with previously identified NucC proteinsaov function in the stats R package. Multiple comparisons between positive samples and negative controls were performed using Dunnett’s test with multcomp R package. Reaction slopes were determined by extracting coefficients from linear models fitted to fluorescence data with lm function in R. The linear regions of the fluorescence curves were identified using rolling regression with auto_rate function in respR package. Statistical threshold for detecting SARS-CoV-2 in patient samples with Csm-based assay was set as mean of negative control ± 2.33S.D., which captures 98% of variation in negative samples . Statistical significance levels used in the figures are ***p < 0.001, **p < 0.01, and *p < 0.05. No statistical method was used to predetermine the sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment. No data were excluded from the analyses.All statistical analyses were performed in RStudio. Analysis of Variance Models (ANOVA) were calculated with Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Telomere shortening rates inversely correlate with life expectancy and hence it is critical to understand how telomere shortening is regulated. Recently, the telomeric non-coding RNA, TERRA has been implicated in the regulation of replicative senescence. To better understand how TERRA is regulated we employed a proteomics approach to look for potential RNA regulators that associate with telomeric sequences. Based on the results, we have identified proteins that may regulate TERRA in both a positive and negative manner, depending on the state of the telomere. In this mini-review, we discuss and speculate about these data to expand our understanding of TERRA and telomere interactors with respect to telomere shortening dynamics. Telomeres make up the terminal structures of linear chromosomes that protect chromosome ends and contribute to safe-guarding genome integrity . Unless Telomeres are transcribed by RNA polymerase II into Telomere Repeat-containing RNA (TERRA) . TERRA eTERRA and R-loop levels inversely correlate with telomere length, suggestive of a regulatory mechanism to ensure low R-loop abundance at normal length telomeres and increased R-loop accumulation at short telomeres. However, even at short telomeres (where stable R-loops promote repair), the hybrids must also eventually be resolved, as hyper R-loop stabilization accelerates senescence rates in yeast . In thisin vitro in the context of wild-type and senescent cells using mass spectrometry. Subsequently, we repeated the experiment in the presence of recombinant RNase A and RNase H, to identify the protein candidates that associate with telomeric-like sequences in an RNA-dependent manner in the context of senescent cells. This was important as TERRA may participate in the recruitment of proteins to telomeres in senescent cells, when TERRA levels are elevated  and helicases are interesting, and obvious, candidates for the regulation of TERRA and telomeric R-loops  and makeRBPs likely ensure balanced R-loop levels at telomeres through the binding of nascent TERRA transcripts, similar to their role at other transcribed loci . HelicasWith regards to helicases, it is particularly interesting that we have identified yeast Upf1 as a telomeric interacting protein. In human cells it has been demonstrated that UPF1 facilitates telomere replication specifically on the leading strand (where TERRA hybridizes) in non-senescent cells . Moreovein vitro .In addition to helicases and RBPs, other telomere binders identified may be implicated in HDR-mediated telomere maintenance in telomerase-null cells, among them the repair factor Mgm101 and the transcription factor Sdd4.Mgm101, one of the strongest hits in the proteomics screen for telomere binders, plays a role in the maintenance of mitochondrial DNA but also participates in the nuclear DNA damage response . InteresThe transcription factor Sdd4 was identified as telomere-associated protein in WT cells . Its expThe identification and characterization of novel telomere factors remains essential to understand how telomere length is regulated. Since TERRA R-loops have been shown to regulate rates of telomere shortening  and sene


Macrostomum lignano, an S-phase kinase-associated protein 1 (SKP1) homologous gene was identified as enriched in proliferating cells, suggesting that it can function in the regulation of stem cells or germline cells since these are the only two types of proliferating cells in flatworms. SKP1 is a conserved protein that plays a role in ubiquitination processes as a part of the Skp1-Cullin 1-F-box (SCF) ubiquitin ligase complex. However, the exact role of Mlig-SKP1 in M. lignano was not established. Here, we demonstrate that Mlig-SKP1 is neither involved in stem cell regulation during homeostasis, nor in regeneration, but is required for spermatogenesis. Mlig-SKP1(RNAi) animals have increased testes size and decreased fertility as a result of the aberrant maturation of sperm cells. Our findings reinforce the role of ubiquitination pathways in germ cell regulation and demonstrate the conserved role of SKP1 in spermatogenesis.In a free-living flatworm,  Macrostomum lignano is a free-living marine flatworm that in recent years has been developed into a potent model organism to study regeneration and a multitude of other biological phenomena [M. lignano is easy to culture in a laboratory, it has a short generation time, small size, and transparent body, and can regenerate most tissues and organs. It is a hermaphroditic organism that reproduces exclusively in a sexual manner [M. lignano biology, including antibody labeling, in situ hybridization (ISH), RNA interference (RNAi), and transgenesis [henomena . M. lignl manner . Numerousgenesis ,9,10,11.M. lignano, which is based on the proliferation of somatic stem cells (neoblasts), is one of the major focus areas for research using this model organism [M. lignano, neoblasts could differentiate into almost any cell type within several days [M. lignano has been generated [SKP1 gene, which was previously identified as specifically expressed in proliferating cells of M. lignano [The regeneration of organism ,12,13. Iral days . Furtherenerated ,12,15,16SKP1) is a core component of the SCF (Skp1-Cullin 1-F-box) ubiquitin ligase complex [Caenorhabditis elegans, it was shown that SKP1-related genes perform critical functions in regulating cell proliferation, meiosis, and morphogenesis [SKP1 is involved in the regulation of double-stranded DNA breaks and spermatogenesis [Schmidtea mediterranea, knockdown of the SKP1 gene leads to regenerative defects, such as the impaired formation of blastema and the brain [M. lignano, an SKP1 homolog (Mlig-SKP1) is also expressed in proliferating cells, but its functions have not been demonstrated. Therefore, we investigated the effects of Mlig-SKP1 mRNA knockdown on M. lignano using RNA interference (RNAi) in order to determine the role of the gene in regeneration and, consequently, its role in neoblast activity. Unexpectedly, we found that the knockdown has no noticeable effect on neoblast activity, but it has a great impact on the animal’s fertility, rendering it sterile in certain conditions. Our results show that the Mlig-SKP1 gene plays a major role in spermatogenesis in M. lignano.S-phase kinase-associated protein 1 , was selected from a previously established list of markers for proliferating cells [Mlig-SKP1 in homeostatic and regenerative conditions. A high efficiency of gene knockdown by soaking animals in Mlig-SKP1 dsRNA-containing medium was confirmed by qPCR  animals would affect their fertility, we performed fertility measurement experiments. For this, adult animals were treated with dsRNA against Mlig-SKP1 for two weeks and then were put together in new experimental wells to cross-fertilize and lay eggs for four days, after which the animals were removed from the wells, and the laid eggs were allowed to develop. One week later, the number of hatchlings in each well was counted and used to assess the fertility of the worms. While control GFP (RNAi) animals on average produced 0.9625 ± 0.2054 progeny per parent, Mlig-SKP1(RNAi) animals produced 0.3125 ± 0.2126 progeny per parent .To investigate whether the changes in testes morphology in r parent   animals, we performed additional RNAi experiments in 3–5-day-old hatchlings. We observed only a slight delay of 2–3 days in the maturation of Mlig-SKP1(RNAi) hatchlings, but otherwise the fertility of these young adult animals was the same as for the animals where knockdown was started only in the adult stage. Interestingly, after the dsRNA treatment was stopped, the fertility in both groups of animals recovered in a period of 1–2 weeks. These observations suggest that Mlig-SKP1 does not play a role in the development of tissues other than gonads, and even there its absence shows only temporal changes.To investigate whether gonad development is affected in Mlig-SKP1(RNAi) animals, we performed microscopy analysis, which revealed that the increased volume of the testes might be a result of an accumulation of aberrant sperm cells. The sperm in Mlig-SKP1(RNAi) animals looks disorganized and sphere-like  sperm cells have only one short tail of spermatids instead of four, and in comparison to the Mlig-sperm1 knockdown, the same disorders (wrong shape of the flagellum and head) seem to be involved.To further characterize changes in the testes of ere-like , suggestlig-elav  and Mligg-sperm1  knockdowMlig-SKP1 gene characterized in this work shows no connection to the regulation of neoblasts. Instead, we found that it plays a role in the spermatogenesis process. The lack of Mlig-SKP1 results in the disruption of sperm maturation, leading to the increased size of testes and decreased fertility.The SKP1, the main role of the SKP1 protein in mammals is being a part of a complex responsible for ubiquitination [SKP1 was also recently demonstrated to play roles in early meiotic processes in mammals [SKP1 is also required for spermatogenesis indicates that this is a deeply conserved function of this gene.According to previous studies of tination ,18. Impo mammals , and in  mammals , by reguMacrostomum lignano [Nitzschia curvilineata ad libitum. In the present study, we used the wild-type NL12 strain [ lignano  flatworm lignano ), at 20 2 strain .M. lignano worms was performed by soaking as previously described [GFP as a control and for Mlig-SKP1 RNAi treatment, respectively. Twenty-day-old worms were maintained in 24-well plates and incubated with 300 μL dsRNA solution (10.0 ng/μL) in f/2 medium containing diatoms. The dsRNA solution was changed daily. In a part of experiments, half of the wells for both Mlig-SKP1 knock down and control worms were amputated above testes after 2 weeks of RNAi treatment. After 3–4 weeks of RNAi treatment, when all amputated worms were recovered, the specimens were examined with Zeiss Axio Zoom V16 stereomicroscope equipped with an HRm digital camera . For the observation of testis components and sperm morphology, live worms were taken randomly and relaxed in MgCl2 7.14% of f/2 medium. Then the testis were directly observed whole and after the testis ruptured using Zeiss LSM 780 inverted microscope equipped with DIC optics.RNAi treatment of escribed ,26,27. WMlig-SKP1 were amplified from cDNA by standard PCR with BioMaster LR HS-Taq PCR (2×), followed by cloning into the pGEM-T-Easy vector system  and sequenced by the SB RAS Genomics Core Facility . Primers are listed in The colorimetric whole-mount in situ hybridization (ISH) with a PCR-amplified DIG-labeled antisense RNA probe was performed according to previously described protocols ,15,27,28Mlig-SKP1 RNAi knockdown on worms’ fertility, the following experiment was conducted: groups of 20 adult worms were placed in a 24-well plate and daily treated with dsRNA, dissolved in freshly prepared f/2 medium, for three weeks. For fertility measurement, the number of hatchlings in each well was counted twice a week. Moreover, 2–3-day-old hatchlings were taken to start a new experiment with the same conditions.To evaluate the effect of Mlig-SKP1 or GFP dsRNA for 2 weeks. After that, each replica was rinsed with a fresh f/2 medium, suspended in 500 μL of TRIzol reagent , and stored at −80 °C. Total RNA was extracted from the samples following the manufacturer’s protocol. The RNA was reverse transcribed to generate cDNA using Reverse transcriptase RNAscribe RT (Biolabmix) with a mix of oligo(dT) and Random Hexamer primers. Expression of Mlig-SKP1 mRNA was checked along with that of 3 housekeeping genes used in previous studies [To confirm the efficiency of the RNAi knockdown, we performed qRT-PCR using 3 replicas of 20 worms, which were treated by either  studies ,27. The M. lignano gene homologous to human SKP1 (Mlig-SKP1). After the knockdown of Mlig-SKP1 by RNAi we observed a phenotype associated with dysfunction of the spermatogenesis process and formation of aberrant sperm, which caused the enlargement of testes and decreased fertility. Recently, a crucial role of SKP1 in mammalian spermatogenesis was demonstrated [SKP1 is required for spermatogenesis in flatworms, we demonstrate that this is an ancient conserved function of this gene.We investigated the functional role of an nstrated ,20,21, a


CHK1 mRNA. Our results present a versatile protein-based RNA manipulation technology using PPR proteins that facilitates the understanding of unknown RNA functions and the creation of gene circuits and has potential for use in future therapeutics.RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins. First, we developed a rapid construction and evaluation method for PPR-based designer sequence-specific RNA-binding proteins. This system has enabled the steady construction of dozens of functional designer PPR proteins targeting long 18 nt RNA, which targets a single specific RNA in the mammalian transcriptome. Furthermore, the cellular functionality of the designer PPR proteins was first demonstrated by the control of alternative splicing of either a reporter gene or an endogenous  The emergence of genome/transcriptome data in the last two decades has greatly facilitated our understanding of biological organisms and their uses. The recent establishment and popularization of genome editing technologies will provide new opportunities for rational genome engineering in various fields ,2,3. DesRNA manipulation can be divided into two classes: nucleotide-based and protein-based. Nucleotide-based technology includes antisense oligonucleotides (ASOs) and RNA interference (RNAi), which have been widely used for knock-down or inhibition of target RNA functions in basic biological research and various therapeutics ,7. HowevHowever, protein-based technologies present promising advantages, as seen in various cases of cellular RNA processing that have been managed by RNA-binding protein factors. The manipulation of a target RNA can be achieved in a desired manner by fusing a sequence-specific RNA-binding protein with an enzymatic effector domain . A well-These disadvantages in protein-based technologies may be resolved by using pentatricopeptide repeat (PPR) protein, which is a sequence-specific RNA-binding protein whose gene family is extraordinarily expanded in land plants . The PPRKD , the other amino acid sequences of natural PPR proteins in plants are highly degenerated. To date, various PPR scaffolds have been tested using consensus or semi-consensus sequences of natural PPR proteins ,26,27,28KD , whereasTo address the possibility of developing designer PPR proteins as part of a new protein-based RNA manipulation technique, we evaluated the characteristics of dozens of designer PPR proteins. High-throughput construction and evaluation systems for designer PPR proteins have been developed for this purpose. With improvement of the PPR scaffold, we have demonstrated that designer PPR proteins with 18 nt RNA recognition can be prepared in a highly successful production ratio —proteins with higher RNA binding affinities and specificities than previous synthetic PPR proteins. Furthermore, the use of designer PPR proteins in splicing control of endogenous mRNA in human cultured cells has been demonstrated. Therefore, the use of designer PPR proteins as versatile protein-based RNA manipulation tools in basic and applied biology is proposed.For the golden gate assembly of the designer PPR protein with 18 PPR motifs, an intermediate plasmid comprising two PPR motifs was chemically synthesized . The sequence information regarding the SCD, PPR2.0, and PPR3.0 scaffolds for the respective base recognitions  is shown in BpiI site (E. coli. The expression vectors (pMgg_sp and pMgg_gfp) were used for the mammalian expression under the control of the CMV promoter.Prior to the golden-gate reaction for PPR motif assembly, extra domain sequences to be fused to the N- or C-termini of the PPR gene were integrated into the expression vector with the piI site . The extBpiI and T4-DNA ligase , 0.5 µL of Quick ligase (New England Biolabs), and 25 ng of the expression plasmid. The reaction was conducted in a thermal cycler, according to the following temperature cycles: 37 °C for 5 min and 16 °C for 7 min, for 15 cycles. After the reaction, 0.4 µL of BpiI was added to the tube and further incubated at 37 °C for 30 min and 75 °C for 6 min. To degrade non-ligated DNA fragments, 0.4 µL of 1 mM ATP and 0.15 µL plasmid-safe nuclease  were mixed and incubated at 37 °C for 15 min. Then, 3.5 µL of the reactions were transformed into XL1-blue-competent cells . To confirm the size of the inserted PPR repeats in the expression plasmid, colony PCR was performed with Fw 5′-tacagggcgcgtcccattcgcca-3′ and Rv 5′-tcactataggggaattgtgagcgga-3′. The amplified fragments were analyzed using MultiNA . Sequence information for the PPR proteins used in this study and their RNA target sequences is shown in The designer PPR gene of 18 PPR motifs was assembled using the pL-1 to pL-9 plasmids and the expression plasmids via a golden-gate reaction with A ligase . Twenty 2, 0.5% NP-40 , 1 mM PMSF, 2 mg/mL lysozyme, and 2 µL 10 mg/mL DNase) and then sonicated. Cell debris was removed after centrifugation . Half of the lysate solution was stored at −80 °C until use. For purification of His-tagged MS2 protein, 200 µL Ni-NTA slurry beads  were added to the lysate, incubated with rotation at 4 °C for 1 h, and washed three times with wash buffer . The recombinant protein was eluted using elution buffer  and was dialyzed at 4 °C overnight in dialysis buffer .The expression plasmids encoding the His-tag fused MS2 protein gene were transformed into Rosetta 2(DE3) . LB culture (100 mL) supplemented with 200 µg/mL ampicillin and 35 µg/mL chloramphenicol was inoculated with 1/100 volume of pre-cultured transformed cells and cultured at 37 °C until OD600 = 0.5. The recombinant proteins were induced with 0.1 mM IPTG at a final concentration of 15 °C for 16 h with shaking. After harvesting and centrifugation, the cell pellets were dissolved in ice-cold extraction buffer  and incubated at 4 °C with rotation for 1 h. The beads were washed three times with wash buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 0.5% NP-40. The protein was eluted using a biotin elution buffer . Ni-NTA beads (200 µL) were added to the eluted protein solutions immediately and incubated at 4 °C with rotation. The buffer conditions for each step, including washing, elution, and dialysis, were the same as those mentioned above. The concentration of purified proteins was determined using the Pierce 660 nm Protein Assay Kit , according to the manufacturer’s protocols.2PO4, 6.7 mM Na2HPO4, 125 mM NaCl, and 5% (w/v) sodium dodecyl sulfate. After blocking, the membrane was incubated in 10 mL blocking buffer including 0.5 µL streptavidin-HRP (ab7403) for 15 min with gentle shaking. The membrane was washed five times with 0.1× blocking buffer and equilibrated with buffer containing 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 10 mM MgCl2. After removing the buffer, Immobilon Western Chemiluminescent HRP Substrate (Millipore) was added to the membranes. Chemiluminescent signals were detected using a ChemiDocTM Touch instrument . The shifted and non-shifted band intensities were determined using Image Lab software . The overall apparent KD value was determined from the concentration of protein at which 50% of the RNA probe was bound as an indication of the RNA binding affinity.The biotinylated-RNA (0.2 pmol—the same synthetic RNA as that used in RBP-ELISA) and the purified protein at the indicated concentrations were mixed with 10 µL reaction buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 1 mM DTT, and 1 mM EDTA). The reaction mixtures were resolved on a 6% polyacrylamide gel in 0.5× TBE buffer. The RNA and proteins in the gel were transferred to a nylon membrane  with 1× TG buffer using a semi-dry blotter. After blotting, the membrane was crosslinked and then blocked in membrane blocking buffer containing 6.4 mM NaHE. coli protein expression system. Soluble proteins were extracted using lysis buffer and purified using Ni-NTA, as described above. After purification, the SUMO-tag was removed by treatment with Ulp1, and dialysis was performed with ion-exchange buffer, including 50 mM Tris-HCl (pH 8.0) and 200 mM NaCl. Protein samples were added to the SP column and eluted by gradually increasing the NaCl concentration from 200 mM to 1 M. The PPR-containing fraction was separated with Superdex 200 increase 10/300 GL  with 25 mM HEPES (pH 7.5), 200 mM NaCl, and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP). The amount of protein in each fraction was determined by measuring the absorbance at 280 nm.The SUMO-His-tag fused PPR2.0 and PPR3.0 proteins for the t24 target were expressed by a pET TM, Thermo Fisher Scientific, Waltham, MA, USA), and penicillin–streptomycin  at 37 °C with 5% CO2. For transfection, HEK293T cells were seeded into 24-well plates at 80,000 cells per well on the day before transfection and transfected with 500 ng RG-6 plasmid and 500 ng of the designer PPR-containing plasmid using FuGENE HD . The medium was changed after 24 h, and the cells were incubated for an additional 24 h at 37 °C with 5% CO2.The splicing reporter assay plasmid for RG-6 was obtained from Addgene (#80167). HEK293T cells were cultured using Dulbecco’s modified Eagle’s medium (DMEM) , 10% fetal bovine serum (FBS) , according to the manufacturer’s instructions. cDNA was generated in a 20 µL reaction mixture containing 5 µL total RNA (500 ng), 0.5 µL 100 µM oligo dT20 primer, 0.5 µL 10 mM dNTPs, 0.5 µL 0.1 M DTT, 0.5 µL 5× reaction buffer, 0.5 µL D.W., and 0.5 µL Superscript III (Invitrogen). The mixture was incubated at 50 °C for 30 min and then heated at 85 °C for 10 min to inactivate the reverse transcriptase. The cDNA was stored at −20 °C until use. To determine the influence of splicing efficiency by rPPR, PCR was performed with RG6Fw (5′-CAAAGTGGAGGACCCAGTACC–3′) and RG6Rv (5′-GCGCATGAACTCCTTGATGAC–3′) for the RG6 reporter assay, 5′-CHK1_F1 (CTCGGTGGAGTCATGGCAGTG–3′) and CHK1_R1 (5′-CATCTTGTTCAACAAACGCTCACG–3′) for the CHK1 exon 3 skipping assay, and MDM2_Ex3_Fw (5′-CGCGAAAACCCCGGATGGTGAG–3′) or MDM2_Ex6_Fw (5′-CATGATCTACAGGAACTTGGTAGTAGTC–3′) or MDM2_Ex7_Fw (5′-GAGAACAGGTGTCACCTTGAAGGTGG–3′) and MDM2_Ex12_Rv (5′-ATTCATTTCATTGCATGAAGTGCATTTCC–3′) for the MDM2 exon 9 skipping assay, using PrimeSTAR GXL DNA polymerase . The PCR product was analyzed using MultiNA  with a DNA 1000 kit . The ratio of exon skipping was calculated as the band intensity of the exon-skipped RNA divided by the sum of the exon-skipped and included RNAs.Total RNA was extracted using a MaxwellFluorescent images were captured using a Leica DMi-8 with FITC, RHOD, and DAPI filters for GFP, dsRED, and DAPI staining, respectively. For the RG-6 reporter assay, to determine the exposure time for imaging, only reporter-transfected cells were used to investigate the conditions under which the fluorescence intensities of FITC and Texas RED were comparable.chk1a and PPRsp6 were cloned into a modified pSBtet-Pur vector . The PPR expression plasmid and pCMV (CAT) T7-SB100  were transfected into HEK293T cells, which were selected using puromycin. Cells (1.5 × 105) from each stable cell line were seeded into 6-well plates, and 0, 1, or 10 µg/mL doxycycline was added one day after seeding. After three days, total RNA was extracted using Maxwell . Exon skipping of CHK1 exon 3 was analyzed by RT-PCR. For analysis of cell viability, after three days of induction with 0 or 10 µg/mL Doxycycline (Dox), cells were collected and cell survival was determined by counting, using a Countess® II FL  after trypan blue staining.SV40 NLS-fused PPRVarious concepts of designer PPR proteins were tested . We begaThe RNA binding selectivity of the PPR motif is determined by amino acid species at the 2nd, 5th, and 35th amino acid positions, known as the PPR code . The VTIn contrast to oligonucleotide-based technology, a disadvantage of protein-based technologies is their complicated construction method, which requires substantial expenditures of time and effort. The construction of proteins with modular structures, including TALE, PUF, and PPR genes, has been achieved using golden-gate cloning techniques ,31,32.The TALEN assembly containing 12 to 20 TALE repeats was conducted using a two-step reaction. First, a pre-step can be avoided by preparing an intermediate consisting of two or four TALE repeats a. The acThe target length of the designer PPR protein was determined with 18 nt recognition for use in mammalian cells, which is the same length as the designer TALE protein and theoretically sufficient to target a single RNA in the mammalian transcriptome. To construct a designer PPR protein containing 18 PPR motifs, we applied a similar golden gate assembly using two PPR motifs as an intermediate to reduce the total number of intermediates. DNA sequences of the 144 PPR motifs (=16 × 9) were chemically synthesized while maintaining degeneracy at the nucleotide level to avoid homologous recombination between DNA sequences encoding PPR motifs .Using this method, four designer PPR protein genes with different target RNA selectivities were constructed using the PPR2.0 scaffold, in addition to the SCD scaffold as a control. The cloning efficiency of the four designed PPR genes of the PPR2.0 or SCD scaffolds was estimated by electrophoresis and sequencing by choosing three clones per PPR gene . The cloEvaluation of RNA binding affinity and selectivity of the recombinant protein provides fundamental information. However, this also requires substantial time and effort, including purification, dialysis, and quantitation, and the use of low-throughput conventional analytical methods, such as electrophoretic mobility shift assay (EMSA) or surface plasmon resonance analysis using Biacore a.To overcome this disadvantage, we designed a new method designated the RNA-binding protein ELISA (RBP-ELISA), which is a 96-well-plate-based ELISA-like assay that can be used to evaluate the RNA binding affinities and selectivities of expressed recombinant proteins using cell lysates without purification. To perform this method, 5′-biotinylated synthetic RNA containing the RNA sequence of interest was immobilized on a streptavidin-coated 96-well plate b. An RNAE. coli as a conjugate of Nano-luc and 6×His-tag. First, the recombinant protein was purified from half of the cell lysate and subjected to RBP-ELISA using the target RNA and non-target RNA . The estimated correlation coefficient between the 70 RBP-ELISA scores and ΔG was low  to cis-elements in pre-mRNA. Artificial control of alternative splicing has been achieved by blocking cis-elements using various techniques, such as ASO and CasRx [Eukaryotic RNA undergoes various types of RNA processing and modifications. Among these, we focused on splicing to address the application of designer PPR proteins in manipulating the RNA of interest in mammalian cells. It is known that 95% of human genes have two or more spliced forms via complexed mechanisms . Alternand CasRx ,33.In this study, an available reporter system, a bi-chromatic fluorescent reporter plasmid designated RG-6, was used . The repThe plasmid of RG-6 and the PPR genes were transiently co-transfected into HEK293T cells, and the splicing pattern change was initially examined by EGFP or dsRED fluorescence using microscopy b. The dsHowever, when the PPR gene was fused to a fluorescent protein gene to analyze cellular localization, unusual distribution or possible aggregation was occasionally observed in the designer PPR proteins of the PPR2.0 scaffold . TherefoWhen the PPR3.0 scaffold was applied to the RG-6 reporter assay, pronounced improvements in exon 2 skipping were observed by microscopy b. The spcheckpoint kinase 1 (CHK1) mRNA as a model molecule. The CHK1 protein is known as a key regulator of the checkpoint signaling in both the unperturbed cell cycle and DNA damage response [CHK1 pre-mRNA is controlled through the cell cycle, and two protein isoforms, CHK1 and CHK1S, are produced in the presence or absence of exon 3, respectively. The highly abundant CHK1S protein inhibits CHK1 function at checkpoint signaling, and overexpression of CHK1S in cancer cells induces cell death [Toward the end of this study, we applied the PPR technique to endogenous mRNA splicing, choosing response . Splicinll death . If accuCHK1 mRNA splicing, the putative enhancer element(s) in exon 3 was computationally searched using a human splicing finder [chk1a than for PPRchk1b -inducible promoter. Two days after Dox induction of the PPRchk1a gene, the splicing extent of CHK1 mRNA and cell survivability were evaluated. RT-PCR analysis revealed efficient exon 3 skipping, as observed in the transient expression of the PPR gene . RT-PCR analysis revealed no splicing abnormality around exon 9 of MDM2 RNA  as well as a single specific RNA in the mammalian transcriptome. Furthermore, we demonstrated applications in splicing control in cultured human cells by targeting endogenous To reduce the time and effort required to construct designer RNA-binding proteins, including designer PPR proteins, we built a 96-well-plate-based construction and evaluation system. Using this rapid system, 100 designer PPR proteins were obtained in six days. This system enables the design, construction, and evaluation of designer PPR scaffolds using hundreds of the test PPR molecules. In addition, acquiring biochemical characteristics prior to analysis in cells or individuals would provide significant advantages with respect to understanding the mechanism, which is difficult for nucleotide-based technologies. In principle, the RBP-ELISA system can be applied for the analysis of other RNA- and DNA-binding proteins.atpH mRNA [This study also exhibited a high production rate (91%) of designer PPR proteins with expected RNA binding affinities and selectivities. Furthermore, we highlighted the wide range of applications of various RNA species, including RNA with high GC content and highly structured RNA, as shown. When the PPR protein binds to the RNA structure, it is expected that the PPR protein dissociates the structure, since the PPR protein is sequence-specific rather than structure-specific. The plant PPR10 protein has been shown to bind and refold to the secondary structure located in the 5′UTR of tpH mRNA . These ctpH mRNA ,12.trans-splicing factors to cis-elements on the pre-mRNA can mediate exon inclusion, exon exclusion, or the creation of alternative splice sites [cis-element dependency. Further analyses will be required to evaluate the efficiency and off-target effects of designer PPR proteins and the nucleotide-based method in parallel, as well as to further understand alternative splicing.Alternative splicing is a hallmark of mammalian transcriptomes. The mechanism remains, but it is widely held that binding of the ce sites . In the ce sites . Indeed,ce sites . This suE. coli [In this study, the sole PPR motif repetition was applied to the splice control, as in ASO. Various modes of action using designer PPR proteins would be expected by fusing the effector protein domain, such as translational enhancement, which we have previously shown using a fusion protein of natural PPR protein of CRR4 and a partial eIF4G domain in HEK293T cells . SimilarE. coli ,44. MostE. coli . Fusing In summary, designer PPR proteins would provide a simple and scalable option to study and manipulate RNA, in addition to the RNA-targeting CasRx system. Although the protein-based tools, including designer PPR proteins, still require complicated procedures for their use compared to the RNA-targeting CRISPR/Cas system, we would anticipate attractive advantages for protein-based tools, as seen in various catalytic reactions in which nucleotides have been replaced with proteins during biological evolution. Further analysis is required to address the advantages and disadvantages of protein-based and nucleotide-based tools and to determine their appropriate usages depending on applications. We hope that the new protein-based RNA engineering tool presents a new frontier in biology, as do genome editing tools, such as zinc-finger, TALE, and CRISPR/Cas9.Kyushu University and EditForce, Inc., have filed patents regarding an efficient method for the construction of the PPR gene (PCT/JP2020/021472) and improvement of the PPR scaffold to increase its solubility (PCT/JP2020/021473). Y.Y., M.T., T.I. and T.N. are the inventors listed in the patents.


The human history has witnessed the rapid development of technologies such as high-throughput sequencing and mass spectrometry that led to the concept of “omics” and methodological advancement in systematically interrogating a cellular system. Yet, the ever-growing types of molecules and regulatory mechanisms being discovered have been persistently transforming our understandings on the cellular machinery. This renders cell omics seemingly, like the universe, expand with no limit and our goal toward the complete harness of the cellular system merely impossible. Therefore, it is imperative to review what has been done and is being done to predict what can be done toward the translation of omics information to disease control with minimal cell perturbation. With a focus on the “four big omics,” i.e., genomics, transcriptomics, proteomics, metabolomics, we delineate hierarchies of these omics together with their epiomics and interactomics, and review technologies developed for interrogation. We predict, among others, redoxomics as an emerging omics layer that views cell decision toward the physiological or pathological state as a fine-tuned redox balance. In other words, “top down” approaches, largely attributable to “omics” development, coupled with “bottom up” strategies to offer a holistic tool for efficient biological system investigation. The concept of dissecting complex disorders including cancers has been, accordingly, advanced from static delineation between cell malignant and heathy states in a low-throughput manner to spatio-temporal dynamic deconvolution of complex systems involving multi-layer modifications at genomic, transcriptomic, proteomic, and metabolic levels in a global-unbiased fashion.Ever since the establishment of the first high-throughput technology, DNA microarray , technolWhile we are running close to the truth in principle, the ever-growing knowledge on cellular omics persistently transforms our understandings toward cell machinery complexity that challenges our goal toward the fully harness of cell pathological state rewiring. It is, thus, time to comprehensively review what has been done and is being done in omics-relevant studies to forecast what can be done in “omics” as a shortcut toward our goal. Focusing on the four big omics, i.e., genomics, transcriptomics, proteomics and metabolics, their epiomics and pair-wise interactomics, this paper comprehensively reviews high-throughput technologies developed, and forecasts, among others, the emerging role of “redoxomics” on the cell machinery.Sequencing and mass spectrometry (MS) are basic experimental tools availing in our tour in investigating the omics of a given biological system. While sequencing-based approaches are feasible for studies on genome, transcriptome, their epitomes and interactomes involving DNA/RNA, MS-based techniques can be used to interrogate proteome, metabolome, and interactomes that do not involve DNA/RNA , Table 1Genomic techniques are dedicated to investigate the inter-individual variations at both the germline and somatic levels via sequencing the genome of interest. The development from DNA microarray technology , first ghttps://www.ncbi.nlm.nih.gov/probe/docs/techbeadarray). The DNA microarray technology is relatively mature, with various well-established experimental platforms and analytical tools available  and high per-base accuracy   , and dom99.999%) , 63. Yet99.999%) , 64.NGS genome sequencing, comprised of primarily four categories, i.e., cyclic-array sequencing , 66, micTGS, the third revolution in sequencing technology as enabled by Pacific Biosciences   and OxfoIt is worth mentioning that, despite the rapid development and increasing popularity of NGS and TGS, the DNA microarray technology still gains favor in genome-wide association studies (GWAS) for the sake of economy, and there is a gaining momentum to combine DNA microarray and NGS/TGS in genotyping toward increased resolution of population-specific haplotypes and imputation strength .Genomic sequencing technologies have been applied to characterize many genetic disorders , long non-coding RNAs (lncRNAs), and circular RNAs (circRNA). Transcriptomics techniques aim to detect and quantify RNA molecules transcribed from a particular genome at a given time .Prior to the advent of NGS, RNA microarray was used as the conventional experimental technique to detect mRNA alterations within cells of interest at different stages in a high-throughput manner. RNA microarrays can be used to profile differentially expressed genes and identify markers capable of distinguishing cells between the normal and cancer states by concomitantly quantifying the relative mRNA abundance of thousands of genes.de novo transcripts can be identified using RNA sequencing techniques given its independence on existing probes. Aided with the RNA sequencing technology, a vast amount and diversified types of non-coding RNAs (ncRNAs) have been discovered and found to be pervasively transcribed from the intergenic and intronic genome regions  such as digital gene expression (DEG) sequencing and 3' end sequencing, sequencing approaches to probe alternative splicing and gene fusion, targeted RNA sequencing, and single cell RNA sequencing  is another tool for whole genome methylation profiling that relies on the precipitation of methylated DNA by the recombinant methyl-CpG binding domain of MBD2 followed by sequencing .de novo DNA modification detection.Illumina short-read sequencing has been coupled with immunoprecipitation for DNA modification detection which, though being feasible for identifying epigenetic alterations in broad genomic regions, cannot reach the resolution at a single base level nor differentiate reads from different cells. Long-read sequencing technologies such as PacBio and Oxford nanopore sequencing techniques have thus been adapted for epigenome interrogation. PacBio SMRT, which monitors the polymerase in real time during DNA sequencing, detects epigenetic modifications by monitoring the inter-pulse durations in the reading rate of a polymerase as a result of kinetic variation . ONT forEpigenomic sequencing technologies have enabled, for example and among others, non-invasive epigenomic molecular phenotyping of the human brain , rapid eEpitranscriptomics seeks to elucidate the role of RNA structure and modifications in regulating gene expression, where RNA modification focuses on modified nucleotides in mRNA .in vitro methods, chemical-based in vitro methods, and chemical-based in vivo methods   to generate a mixture of RNA fragments that, once been analyzed by sequencing, allows for RNA secondary structure inference. Chemical-based methods utilize small membrane permeable molecules such as nucleobase-specific chemicals, carbodiimide modifying reagents, and ribose-specific probes to interrogate RNA structure, which can be applied both in vitro and in vivo and often capable of achieving single-nucleotide resolution. Chemical-based in vitro methods include structure-seq  (in vivo click selective 2-hydroxyl acylation and profiling experiment (icSHAPE) (in vivo (MARIO) (Sequencing-based methodologies for mapping RNA structures can be classified into enzyme-based  methods . Enzyme-e (PARS) , fragmene (PARS) , parallee (PARS) , and proe (PARS) . These mture-seq , dimethyture-seq , and higture-seq . ChemicaIRS-seq) , selectiIRS-seq) , in vivoicSHAPE) , and map (MARIO) . CombiniWhile chemical modifications on RNA are evolutionarily conserved traits of structural RNAs such as rRNA and tRNA , their pThe 13 chemical modifications identified so far in mRNAs can be divided into two categories, i.e., modifications of nucleotides adjacent to the 5' cap and internal RNA modifications. Cap-adjacent nucleotide modifications typically regulate RNA stability and translation, and can occur in mRNA, primary miRNA transcript, lncRNA, small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA) . InternaAs the abundance of mRNA modifications is in general low, large amounts of mRNAs are needed or very deep sequencing is required using current experimental approaches. This has catalyzed the generation of many sequencing-based approaches for transcriptome-wide mapping of mRNA modifications. The most prevalent approach is RNA immunoprecipitation (RIP) sequencing (RIP-seq), where modified nucleotides are recognized by antibodies followed by whole transcriptome sequencing (RIP-seq) . Other aBesides, using chemical reactions specific to a given RNA modification followed by short read sequencing provides an alternative approach for RNA modification detection. For instance, RNA bisulfite sequencing (RNA-BisSeq) is one of these approaches that relies on chemical deamination of cytidine to uridine by sodium bisulfite, leaving m5C intact –153. A sEpitranscriptomic sequencing technologies have enabled us to profile the landscape of epitranscriptomic RNA modifications  toward eGiven the prevalence of DNA-RNA interactions at the transcription start sites , 159, diMethods fell into the first category include chromatin isolation by RNA purification (ChIRP) , captureTechnologies of the second category include mapping RNA–genome interactions (MARGI) , chromatde novo targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs participating in co-transcriptional RNA processing , and psoralen cross-linking . The disric RNAs . Identifric RNAs  and crosric RNAs . Yet, boin vivo RNA crosslinking, RNA fragmentation, intramolecular ligation and reverse crosslinking. It is noteworthy that the choice of crosslinking reagents may determine the types of RNA interactions to be discovered. For example, psoralen or its derivatives as used in PARIS, SPLASH, and LIGR-seq intercalate in RNA helices and can be used to identify hybridized RNA pairs.Methods established for high-throughput RNA interactome analysis leverage proximity ligation to produce chimeric sequences, which include, e.g., psoralen analysis of RNA interactions and structures (PARIS) , sequencInterrogating the RNA-RNA interactome has enabled us to construct the higher-order transcriptome structure of living cells that guided the discovery of lncRNA structures and functionalities , has aidInteractions between proteins and DNA play fundamental roles in transducing genetic information into functionalities. Methods for characterizing such interactions include electrophoretic mobility shift assays, DNase footprinting, ChIP, and systematic evolution of ligands by exponential enrichment (SELEX) –177 whicde novo protein-DNA interactions urgently called for.Protein binding microarrays have also been adopted to investigate transcription factor (TF)-DNA interactions; yet, the binding sites of many TFs are longer than those that can be combined to the array . ChIP-hyChIP-Chip, also known as the genome-wide location analysis, combines chromatin immunoprecipitation (ChIP) and DNA microarray analysis to identify protein-DNA interactions occurring in living cells . It has The pervasive transcription of the genome creates the diversified reservoir of ncRNAs. Despite our limited knowledge regarding the functions and regulatory mechanisms of ncRNAs, it has been well-accepted that interactions between RNA binding proteins (RBPs) and RNAs play vital roles in cell homeostasis maintenance. The dynamic combination, competition and coordination between RBPs and RNA offer many pointcuts to the mechanisms of action of these RNAs. Many experimental techniques have been established to systematically investigate such interactions. High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation, namely HITS-CLIP or CLIP-seq , as wellWhile these technologies have been successfully applied to decode miRNA-target interactions , identifProteomics investigates the functional relevance of all expressed proteins in a cell, tissue or organism [namely proteome ] by inteMS is a primary protein characterization technique that can be used to determine the amino acid sequence of a protein and PTM sites. MS measures charged molecules based on their mass-to-charge ratios, where the mass analyzer characterizes molecules by their mass-to-charge ratios, and the signal intensities of charged peptides reflect the quantity. One significant advance brought by MS to the omics field is the rate at which it identifies proteins in an entirely discovery-driven way. The advent of the high-resolution “LTQ™Orbitrap™ ” MS instThere are several types of commercially available mass spectrometers. These include high-resolution MS methods such as LTQ™Orbitrap™ , matrix-de novo protein characterization and isobaric peptides differentiation (Various tandem mass spectrometric techniques (MS/MS) approaches have been established to generate unique fragmentation profiles from different proteins that enables ntiation . In MS/Mntiation , electrontiation , electrontiation , and colntiation . It is ontiation .Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) are MS coupled with chromatography-based separation modules that convey several advantages over MS. One critical benefit of using LC or GC before MS is the alleviation of the matrix effect and ion suppression by separating analytes from interfering endogenous compounds such as salts and ion pairing agents using an appropriate chromatography. Also, peptides can be quantified by measuring the area under the chromatographic peak. LC-MS has been used to characterize various kinds of proteins with broad ranges of physicochemical properties and molecular weights, and the use of GC-MS in protein characterization is rather rare that has been limited to profile relatively more volatile molecules with lower molecular weights such as protein adducts.Ophiocordyceps sinensis at different culture periods  participating in cell endogenous metabolism, function as energy sources, signaling molecules, and metabolic intermediates with protein modulatory roles (see section on epiproteomics) in complex biological systems. Metabolites have been demonstrated to serve as important biological modulators across multilayer omics toward the maintenance of cellular homeostasis. Metabolome is referred to as a collection of all metabolites in a cell that encompass all biomolecules except for the genome, transcriptome, proteome, and metals.Methods for metabolome interrogation include Fourier transform-infrared (FT-IR) spectroscopy , Raman sFT-IR, Raman and NMR spectroscopies provide non-destructive and rapid solutions for metabolite analysis, where absorption spectra at specific wavelengths determines the structure of unknown metabolites and the area under the curve (AUC) of the absorption spectra quantifies the amount. However, these approaches do not have sufficient sensitivity and selectivity –215.MS is the most feasible tool to probe the metabolome that can detect a wide spectrum of metabolites . Isobaride novo target identification by charactering as many metabolites as possible in a biological sample  prior to MS analysis, “top-down” that defines the proteomes present in a sample through analyzing intact proteins, and “middle-down” that is designed for analyzing histone PTMs as histone N-terminal tails can be cleaved off by specific proteases to generate polypeptides with accessible size for MS detection. The “top-down” approach in a high-throughput fashion can depict a comprehensive and accurate view on the epiproteome of the targeted system, but is challenging as larger molecules are difficult to be separated by LC and analyzed by MS, and the chance of having isobaric proteomes  increases with the portion of protein analyzed .Phosphoproteome is perhaps the most pervasive PTM landscape, the analysis of which adopts the “bottom-up” approach. Peptide digests are typically enriched , fractioned off- or on-line (by chromatographic separation techniques such as reversed phase high performance liquid chromatography) prior to mass spectrometry analysis . A recenEpiproteome interrogation has been largely applied to capture the dynamic phosphoproteome profile of a particular biological system such as prostate cancer , human uProtein-protein interactions (PPIs) represent the most prominent and well-studied molecular interactions within cells. The earliest high-throughput technology employed for PPI identification is yeast two hybrid (Y2H) screening, which works by separating two functional domains of a single TF that elicits signals once brought into close proximity . Y2H scrA high-throughput screening approach has been established to characterize PPIs directly from cell-free protein synthesis reactions. Proteins of interest are immobilized non-covalently on the donor and acceptor beads that produce chemiluminescent signals on interactions. This technique relies on the Amplified Luminescent Proximity Homogeneous Linked Immunosorbent Assay (AlphaLISA) that enables rapid PPI characterization without the need for protein purification and a highly parallel and miniaturized workflow taking advantages of robotic and acoustic liquid handling. This recent technology can characterize competitive binding of proteins for specific epitopes besides direct PPIs, and thus has been applied to screen candidate antibodies capable of competing with the spike receptor-binding domain (RBD) of SARS-CoV-2 for human angiotensin-converting enzyme 2 (ACE2) .Protein-metabolite interactions are essential in maintaining cell homeostasis under the conventional state, and coordinating responses to internal or external stress or perturbations. Protein-metabolite interactions are prevalent in cells which were estimated to be at the scale of millions –242.de novo protein-metabolite interactions was proposed, under the hypothesis that the binding of a metabolite to a protein of interest can block its Proteinase K cleavage sites , is comprised of trillions of microbes inhabiting inside the human body and interacting with their host  and false positive (FP) rates. The reproducibility of MS-based omics is, in general, lower than sequencing-based omics. For example, the results of some MS-based omics such as proteomics, metabolomics and their epiomics are highly dependent on the type of mass spectrometer, as well as the sample processing protocol and data processing pipeline used. This renders the compromise of the accuracy and reproducibility of some aforementioned omics unavoidable in pursuit of the global landscape. Domain specific languages such as Nextflow  have beeAs we learn more about known cell components, novel types of biological players are being discovered that further complicate our existing knowledge on how human cells behave under physiological and pathological conditions. Besides, the number and variety of cells comprising the system to be studied regarding human health has been expanded by an order of magnitude by microbiome . These tAnother important factor contributing to the huge complexity of cells that challenges translational omics is the multi-layer heterogeneity. Omics technologies at a particular level can only present one picture of a cellular system that dynamically transits among varied states. In addition, cells presented in tissues or biopsies can be very heterogeneous, rendering accurate interpretation of omics information challenging. Further, differential clinical manifestations and treatment responses among patients add an additional layer of complexity regarding the clinical use of omics technologies.Accordingly, there had been a trend of integrating multiple omics information toward improved understanding of a particular knowledge domain as evidenced by the generation of immunomics and microbiomics. This enables us to portrait a systematic view on a research area that gains additional value if the topic conveys systematic impacts on human cells regarding its decision toward either homeostasis maintenance under the healthy state or running into a chaotic state that drives cells malignant.Redox imbalance has been indicated as a causal factor of a variety of disorders such as cancer –265 and By leveraging multiplexed fluorescence, DNA, RNA and isotype labeling, and taking advantages of multiomics data interrogation, “spatial omics” has emerged to enable the detection of variations in, e.g., transcriptome and proteome, within their native spatial context  and beenLast but not the least is the critical contribution of bioinformatics to omics interrogation. Various toolboxes have been made available for, e.g., data quality control, pre-processing, abnormal molecule identification, interaction prediction, enrichment analysis, pathway analysis, network construction, as well as more advanced or focused analyses such as pseudo-time and trajectory inference in single cell sequencing. Besides, numerous databases and computational tools have been developed to allow for the easy access and analysis of omics data. The Cancer Genome Atlas (TCGA)  and GeneXD conceptualized the idea, drafted the paper, provided financial support, and final proved the manuscript. XD and LS conducted literature searching and prepared figures and tables. All authors agree with the content and its publication.This study was funded by the National Natural Science Foundation of China (Grant No. 81972789) and Fundamental Research Funds for the Central Universities (Grant No. JUSRP22011).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.