The intrinsic properties of RNA and DNA biopolymers emphasizedby engineered nucleic acid nanoparticles (NANPs) offer accelerateddevelopment of next-generation therapies. The rational design of NANPsfacilitates programmable architectures intended for regulated molecularand cellular interactions. The conventional bottom-up assembly ofNANPs relies on the thermal annealing of individual strands. Here,we introduce a concept of nuclease-driven production of NANPs whereselective digestion of functionally inert structures leads to isothermalself-assembly of liberated constituents. The working principles, morphologicalchanges, assembly kinetics, and the retention of structural integrityfor system components subjected to anhydrous processing and storageare assessed. We show that the assembly of precursors into a singlestructure improves stoichiometry and enhances the functionality ofnuclease-driven products. Furthermore, the experiments with immunereporting cell lines show that the developed protocols retain theimmunostimulatory functionality of tested NANPs. The presented approachenables exploitation of the advantages of conditionally produced NANPsand demonstrates that NANPs’ stability, immunorecognition,and assembly can be regulated to allow for a more robust functionalsystem. Although themolecular rearrangement of this system is spontaneous, cells maintainsubstantial control over the molecular associations.3 The working principles of these active structures havealready inspired the development of responsive, shape-switching proteinand nucleic acid-based nanosystems.6Self-assembly is a ubiquitous naturalprocess wherein moleculesspontaneously organize themselves in higher-order structures basedon noncovalent interactions.7 Significantly, the immunomodulatory properties of NANPs also differfrom the unassembled monomers and can be rationally designed to illicitexpected immune responses that depend on NANPs’ architecturesand routes of delivery.10The self-assemblingnucleic acid nanoparticles (NANPs) are an advancedclass of biomaterials comprised of programmable combinations of structuraland functional RNA and DNA motifs embedded within the same uniquearchitectures. Assembled NANPs often gain new physicochemical propertiesthat can be quite distinct from their original constituents but stillretain the functional roles of nucleic acids.11 The correctly assembled 3D structures of NANPsplay a prominent role in the spatial organization of functional moietiesand define their immunomodulation.13 While widelyused, the high level of technicality of the existing assembly protocolsand the lack of control over the triggered formation of NANPs at isothermalconditions preclude their broader biomedical applications.The conventional bottom-up assembly of NANPsis typically a multistepprocess that requires combining individually synthesized and characterizedconstituent strands, followed by different annealing protocols thatare specific to individual NANPs’ design and composition . Interestingly, the prolonged incubationof up to ∼5 h at 37 °C does not improve the DNA cubes’assembly efficiencies (Supporting Information (SI) Figure S1b), suggesting a relatively quick digestion of hybridduplexes by RNase H and reaching the plateau phase of assembly. Theextended storage at 4 °C maintains the high yields of DNA cubeassemblies . When the incubationwith RNase H is carried out at 4 °C, the decreased digestionrates slow down the formation of DNA cubes . Importantly, no cubes are observed during the extendedincubation of RNA/DNA duplexes without nucleases. It should also benoted that DNA cubes show less aggregation remaining in the loadingwells compared to cubes prepared by the conventional assembly protocol.This could be attributed to the continuous release of individual monomersthat lowers their concentration during the assembly that occurs atthe DNA cubes’ melting temperature.6 Initial assessment of assembly dynamics shows that RNase H-drivenassembly of DNA cubes starts after ∼5 min, and all monomersare assembled in cubes after ∼10 min . Upon closer analysis using 1 min intervals, itis revealed that all DNA cubes are formed at ∼6 min, and theamount does not increase significantly thereafter .The DNA cubes are formed within the minutes afterRNase H addition(S3a). Comparedwith the control assembly, the proportion of RNA materials trappedin the native-PAGE gel wells is higher. There is also no observeddependence of RNA cube assembly yields on storage at 4 °C . Interestingly, RQ1 DNase-drivenassembly of RNA cubes can only be achieved at 37 °C with no cubeformation observed after incubation at 4 °C for 5 h .RQ1 DNase-driven assembly ofRNA cubes requires a significantlylonger time with some RNA cubes only appearing after ∼30 minof incubation. However, the presence of RNA/DNA hybrid duplexes canstill be observed for up to 1 h 1d, S3a. Further comparative analysisof nuclease-driven assembly kineticsshows that RQ1 DNase is significantly slower in degrading hybrid duplexes(∼30 min) and releasing ssRNA strands, while RNase H digestsits RNA substrates in RNA/DNA hybrids at much faster rates (∼10min) 1d,e.17 and the short linear RNA/DNA hybrids are no longer present, indicatingthat the nucleic acid materials are successfully restructured afterthe nuclease digestion.In addition to electrophoretic mobility shift assays, the homogeneityof resulting NANPs and the nuclease-driven changes in morphology fromRNA/DNA duplexes to cubes are confirmed by atomic force microscopy(AFM) 2. While18 SpeedVac is one ofthe simplest available laboratory techniques with the most promisefor future upscaling and cost-effectiveness when addressing storagepossibilities outside the cold chain. However, when compared to otherdehydration techniques ,SpeedVac was shown to have the most negative impact on the structureand recovery of RNA and DNA cubes.Currently, the standard storage and transportation of NANPs in solutionrequire a cold chain that is costly and, under some circumstances,challenging. Our previous study shows that various NANPs retain theirstructures differently when subjected to dehydration methods and subsequentstorage conditions.Figure S4a). Possible harsh conditionsduring shipping and storage outside the cold chain are simulated bystoring the representative samples at +50 °C for 24 h. UnlikeDNA cubes, the integrity of RNA cubes, incubated at +50 °C bothin solution and after dried by SpeedVac, is lost and cannot be recoveredafter rehydration. As expected, RNA/DNA hybrid duplexes resist elevatedtemperatures both in dehydrated conditions and in solution and canbe readily converted to either RNA or DNA cubes after treatment withcorresponding nucleases .The advantage of the presented“break to build” approach is that from one compositionof simple precursor structures, two different NANPs can be produced.Also, RNA/DNA hybrid duplexes are expected to remain intact when driedby any dehydration method available. Therefore, we investigate howthe SpeedVac dehydration affects the storage and nuclease-driven assemblyof NANPs  and luciferase, respectively . For HEK-Lucia RIG-Ireporter cells, none of the transfected DNA cubes elicit RIG-I stimulationcompared to hybrid RNA/DNA duplexes  and all RNA cube assemblies trigger a significantly higher RIG-Istimulation than DNA cubes. Interestingly, the most immunostimulatoryresponses arise from RNA cubes produced from RNA/DNA fibers .Various NANPs have been tested to induce the activation of relevantpattern recognition receptors (PRR). We examined the immunostimulatoryproperties of our assemblies in reporter cell lines that express TLR7(Toll-like receptor 7) or RIG-I (retinoic acid-inducible gene I).TLR7 is an endosomal receptor sensing ssRNAs and short dsRNAs in NANPstructures, while RIG-I is a cytosolic receptor that recognizes RNANANPs with triphosphate on the 5′ ends.ectively 3e,f. Connalogues 3e, S6a. duplexes S6b and a23 This finding elucidates the mechanism underlyingthe faster rate of DNase-mediated digestion of dsDNA compared to theRNA/DNA complex.We employ MD simulationsto investigate the stability and dynamics of RNA structures at twodifferent temperatures, 37 and 45 °C. Our findings 4a,b indi24 and results in much slower release of the ssRNAstrands. Furthermore, we speculate that the incubation temperaturebelow the melting temperature of the RNA cube may allow misfoldingand formation of undesired secondary structures with larger aggregates,which can also occur due to the lower rates of DNase digestion andRNA release. In addition, partially cleaved DNA strands that are stillhybridized with their RNA counterparts may hinder the intended intermolecularinteractions needed for the formation of RNA cubes.The nuclease-driven assembly of NANPs isbased on degradation ofthe inert precursors made of either short RNA/DNA duplexes or longerRNA/DNA hybrid fibers. We show that the addition of either RQ1 DNaseor RNase H affects the dynamics and yields of the subsequent assemblyof NANPs. While the RNase H has evolved to efficiently recognize andcleave RNAs in RNA/DNA hybrid structures, the slower degradation ofhybrids by DNase stems from its preference for the B-form helix, characteristicfor dsDNA, rather than the A-form analogue, presented in RNA/DNA hybrids.Under these conditions, DNase residual activity is less than two percent28 However, this approach requires the presence of cognate NANPs, andthe participating strands can interfere with the assembly processand alter the functionality of the products. Comparatively, in ourmethod, all of the “masking” material is degraded, thuspreventing any hampering in the assembly and function of NANPs.An alternativeapproach for conditional assembly of NANPs is theapplication of strands that engage in toehold interactions with opposingstrands, resulting in the branch migration and liberation of individualstrands.Nuclease-driven assemblies were shown to have similar immunostimulatoryproperties to their counterparts that are prepared by a standard annealingprotocol. While DNA cubes are not immunostimulatory, RNA cubes producedfrom digestion of RNA/DNA hybrids elicit an immune response similarto the control RNA cubes. Interestingly, RQ1 DNase-driven RNA cubesmade from RNA/DNA fiber precursors induce the strongest responses,significantly higher than the one from control RNA cubes. The explanationof increased stimulation requires future investigation but may likelybe caused by uncharacterized byproducts of partial DNA cleavage.30 Therefore, the further development of the “break to build”method, besides the streamlining of NANP assembly, may result in thedesign of nanoparticles that will assemble intracellularly by usingcellular enzyme machinery.In summary, we described a new one-stepprotocol for the conditionalnuclease-driven assembly of either RNA or DNA cube NANPs with distinctimmunostimulatory profiles. Nuclease-driven formation of NANPs onlyrequires physiological temperature and the addition of substrate-specificnucleases. Together with the temperature stability of source materials,our approach allows storage and assembly independent of cold chainand complex laboratory equipment. We also suggest and explore a modethat might be a potential solution to enhance the stoichiometry ofbuilding materials, thus increasing the efficiency of full cube assembly.A possible way would be an arrangement of individual monomer strandson one scaffold. Although only a mono-directional approach of degradation(RNA monomers on DNA scaffold) was investigated, we believe that itcould be engineered for bi-directional modes in the future. Indeed,complexing all monomers into one long RNA/DNA fibers functionallyoutperformed other constructs. Processing of simple nucleic acid-basedtherapeutics by cellular enzymes such as Dicer or RNA ligase has alreadybeen described to enhance the functionality of the delivered construct.SI). PCR was used to amplify RNA cube strand transcription templatesusing MyTaq Mix (Bioline). The PCR products were then purified byDNA Clean and Concentrator kit (Zymo Research). RNA cube monomerswere transcribed by in vitro runoff transcription with T7 RNA polymerasein 80 mm HEPES-KOH (pH 7.5), 2.5 mm spermidine, 50 mM DTT, 25 mm MgCl2,and 25 mM each rNTP at 37 °C for 3.5 h. RQ1 RNase-free DNase(Promega) was used to degrade DNA templates before RNA purificationby 8 M urea PAGE 8%AA (29:1). Excised bands were cut and overnight-elutedin crush and soak buffer . Eluted RNA strands were mixed with 100% EtOH in a 1:1.5ratio and placed in −20 °C for at least 4 h. The mixturewas centrifuged at 14,000 rcf for 30 min. Pellets were rinsed threetimes with 90% ethanol. Samples were dried by SpeedVac at 55 °C,and RNA pellets were resuspended in endotoxin-free water. Absorbanceswere measured using a NanoDrop 2000. NANPs were assembled at an equimolarratio of each monomer. DNA or RNA monomers were mixed and denaturedat 95 °C for 2 min, transferred to 45 °C, and incubatedfor 2 min; after that, assembly buffer  was added, and solution was additionally incubatedat 45 °C for 20 min. Six duplexes were individually preparedby the incubation of complementary RNA and DNA strands at 95 °Cfor 2 min, followed by the addition of the assembly buffer and incubationat room temperature for 20 min. RNA/DNA fibers were assembled by firstassembling individual duplexes and then mixing them in an equimolarratio to a single solution and allowing to assemble for 5 min at 37°C. Assemblies were confirmed either on agarose gels or on an8% (37.5:1) non-denaturing (native-PAGE) polyacrylamide gel with runningbuffer containing 89 mm tris-borate (pH 8.2) and 2 mm MgCl2. The gelswere run in a Mini-Protean electrophoresis apparatus at 200V for 45min and stained with ethidium bromide for 5 min.All DNA templates and primers for PCRsand monomers for DNA cubes and fibers were purchased from IDT (thelist of sequences is available in the AFM imaging was carried out on a freshly cleavedmica surface submerged in an aqueous solution of APS (10(3-aminopropyl)-silatrane)by a MultiMode AFM Nanoscope IV system (Bruker Instruments) in tappingmode. Images were processed by the FemtoScan Online software package.The 5% (v/v) of either RQ1 DNase(Promega) or RNase H (Promega or New England Biolabs) were added tothe mixture of six RNA/DNA hybrid duplexes based on the total volumeof the sample, while 5% of ddiH2O was added instead of nuclease forcontrol. Samples were incubated at 37 °C at different time points.Samples were aliquoted at selected time points and stored at 4 °Cuntil ready for loading on gels.Quantification of band intensities was performedusing ImageLab software version 6.0.1. Lanes and bands were manuallydefined. Kinetics were graphed using OriginPro 2023, and the sigmoidalfit was performed using a Boltzmann function.An IR vacuum concentrator (Labconco)was used to dry all samples at 55 °C and with IR radiation toobtain dehydrated duplexes, fibers, and cubes. Sample tubes (bothdried and in solution) were placed on a heat block at 50 °C for24 h. Samples were resuspended back to the original volumes usingendotoxin-free water and analyzed by native-PAGE.2, areplated at 10,000 cells per well for RIG-I and 40,000 cells per wellfor HTLR7 in a 96-well plate 24 h prior to transfection of all samplesusing Lipofectamine 2000. 10 ng/mL 3p-hpRNA for HEK-Lucia RIG-I or2 μg/mL R848 for HEK-Blue hTLR7 is used as a positive controlfor the indicated cell line. Addition of QUANTI-Blue solution to theHEK-Blue cell line or QUANTI-Luc solution to HEK-Lucia RIG-I cellline following the manufacturer’s protocol gave results forSEAP production or luciferase activity under the effect of 24 h treatment.A Tecan Spark plate reader was used to read the absorbance at 638nm for QUANTI-Blue after 4 h of incubation at 37 °C. For QUANTI-Luc,the plate was read immediately for luminescence with a 100 ms readingtime.HEK-Blue hTLR7 and HEK-Lucia RIG-Icells, purchased from InvivoGen and maintained according to the supplier’sguidelines in an incubator at 37 °C and 5% CO33 The structure of the DNase I–DNA complex (PDBID: 2DNJ) was used as a template to build the initial structure ofDNase/dsDNA and the DNase/RNA/DNA complex. All of the structures wereprepared using the teLeap module in Amber 18. The molecules were solvatedin an octahedral box containing TIP3P water molecules, with a distanceof 9Å maintained between atoms and the box boundary. We addedpotassium ions to neutralize the system, followed by additional K+ and Cl– ions to achieve a concentrationof approximately 0.3 M. We conducted several energy minimization stepsbefore the actual simulation. First, we employed the steepest descentmethod for 1000 steps, followed by 1000 steps using the conjugategradient method to perform energy minimization of the entire system.During the MD simulation phase, we initially applied weak restraintsto the molecules and allowed the system to heat up from 0 K to thedesignated temperature (37 or 45 °C) over 200 ps. Afterward,we removed the restraints on the molecules and performed explicitsolvent MD under constant pressure using the isothermal–isobaric(NPT) ensemble with a time step of 2 fs. The length of hydrogen bondswas constrained using the shake algorithm, while the temperature wasmaintained, and pressure was kept constant at 1 bar throughout thesimulation.The MD simulations were conductedusing the pmemd.cuda program within the Amber 18 software package.We utilized the Amber RNA OL3 force field and protein ff14sb forcefield, which has been previously demonstrated to yield satisfactoryresults when studying the stability and dynamics of RNAs. The startingstructures of RNA-only and RNA with digested DNAs were generated usingthe iFoldRNA program.


Dianthus caryophyllus (carnation) leaves. The CarSV-1 genome has notable sequence similarity (62%) to the well-studied CarSV viroid-like RNA and comprises the complete hammerhead consensus sequences involved in self-cleavage. CarSV-1 co-occurs with carnation viruses, such as CarMV.Here, we report the genome sequence of a new circular viroid-like RNA (CarSV-1) derived from  Carnation mottle virus (CarMV+), and two plants were uninfected , without a protein coat . Small vinfected . The preinfected . All invinfected  and siRN2 leaf fragments into powder using a mortar and pestle and dissolving the powder in QIAzol lysis reagent (Qiagen) for RNA isolation and small RNA enrichment using the miRNeasy minikit (Qiagen). Barcoded small RNA transcriptome sequencing (RNA-seq) libraries were generated using the Ion total RNA-seq kit v2 and Ion Xpress RNA-seq barcoding kit (Thermo Fisher Scientific) and sequenced . Adapter and quality trimming were performed using Torrent Suite v5.8.0 software (default parameters), resulting in 3.3 to 5.3 million reads per sample   to the CarSV sequence and had an identical GC content (51%). Furthermore, it contained the complete hammerhead consensus sequences for both the plus and minus RNA strand forms  47. This snd forms  ; 8034 47. GTGTAGATTCCAGGCTACGGC; reverse primer, TGCCACGTAGCCTCAAGG; probe, TTGGGTCCTCGATGATGGGA) confirmed the presence of CarSV-1 RNA in the CarMV+ samples, with threshold cycle (TC) values of 16 to 18 cycles, as has been observed for CarSV (Carnation etched ring virus (CERV) (Alternanthera mosaic virus (AltMV), based on the observed co-occurrence in this study.Reverse transcriptase quantitative PCR (RT-qPCR) analysis using a CarSV-1-specific primer/probe combination  , 9, CarSPRJNA885393. The genome nucleotide sequence of CarSV-1 has been deposited under GenBank accession number OP506135.The raw sequence reads have been deposited in the NCBI Sequence Read Archive under BioProject accession number


Background: The CRISPR/Cas13a system offers the advantages of rapidity, precision, high sensitivity, and programmability as a molecular diagnostic tool for critical illnesses. One of the salient features of CRISPR/Cas13a-based bioassays is its ability to recognize and cleave the target RNA specifically. Simple and efficient approaches for RNA manipulation would enrich our knowledge of disease-linked gene expression patterns and provide insights into their involvement in the underlying pathomechanism. However, only a few studies reported the Cas13a-based reporter system for in vivo molecular diagnoses.Methods: A tiled crRNA pool targeting a particular RNA transcript was generated, and the optimally potential crRNA candidates were selected using bioinformatics modeling and in vitro biological validation methods. For in vivo imaging assessment of the anti-GBM effectiveness, we exploited a human GBM patient-derived xenograft model in nude mice.Results: The most efficient crRNA sequence with a substantial cleavage impact on the target RNA as well as a potent collateral cleavage effect, was selected. In the xenografted GBM rodent model, the Cas13a-based reporter system enabled us in vivo imaging of the tumor growth. Furthermore, systemic treatments using this approach slowed tumor progression and increased the overall survival time in mice.Conclusions: Our work demonstrated the clinical potential of a Cas13a-based in vivo imaging method for the targeted degradation of specific RNAs in glioma cells, and suggested the feasibility of a tailored approach like Cas13a for the modulation of diagnosis and treatment options in glioma. Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) platform has been set up to screen, identify, and analyze the most effective collateral cleavage sites in reporter RNAs for Cas13a-mediated cleavage and subsequent amplification of the nucleic acid sequences for in vitro detection. The Cas13a-based diagnostic methods have been successfully applied to detect several deadly viruses in humans, including avian influenza A (H7N9), Ebola, hepatitis B (HBV), and most recently, the Severe Acute Respiratory Syndrome-associated CoronaVirus-2 (SARS-CoV-2) Rapid read-out of specific genome sequences with the highest precision, especially at the single nucleotide level, is a prerequisite for genetic screening and molecular diagnosis of human diseases, surveillance of infectious diseases, and genetic research  as well. Despite the availability of cutting-edge next-generation sequencing (NGS) techniques capable of differentiating between allelic variants and retrieving structurally complex repeat sequences, there are trade-offs in terms of specificity, sensitivity, cost, efficiency, and simplicity uenza A HN9, EbolaIDH1) gene mutation is the most common genetic factor in glioma pathogenesis, occurring in 80-90% of grade II and III gliomas and 80% of secondary glioblastomas IDH1 gene mutations play crucial roles in the diagnosis and prognosis of glioma Glioma is the most common and highly malignant primary tumor originating in the central nervous system (CNS). Despite the availability of comprehensive clinical and surgical interventions, including chemo/radiotherapy, conventional cancer therapeutics remain ineffective for almost all glioma cases, with unsatisfactory outcomes, poor prognosis, and recurrent tumorigenesis. The development of targeted therapies involving novel biopharmaceuticals and precise diagnostic and therapeutic tools is a critical challenge to the effective management of glioma patients in the clinic. Notably, the oncogenic gene mutations not only trigger tumorigenesis and cancer metastasis but also directly influence the treatment outcome in glioma patients. Therefore, the development of an accurate diagnosis for early detection and targeted gene/cell therapies for the underlying genetic variations may provide new hope for the effective clinical management of glioma. The isocitrate dehydrogenase 1  .All 2. LwCas13a, IDH1mut, and F3-T3 packaged lentiviruses were gifted by GENECHEM . Puromycin  was used to select IDH1mut-positive cells, which were subsequently transfected with Cas13a for at least 48 h, according to the manufacturer's instructions. To avoid loss of the target gene, transfected cells were readily employed in tests within a month.Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum  was used to culture human GBM cell line U87. The primary cell line TBD0220 was cultured in DMEM/F12 supplemented with 10% FBS. All cells were grown at 37 °C in a humidified incubator with 95% air and 5% COab initio free docking; (4) optimize the 3D-structure of the Cas13a-crRNA complex using GROMACS; (5) predict the secondary structure of target gene fragments using RNAfold; (6) construct the 3D-structure of the target gene fragment using 3dRNA; (7) construct the 3D-structure of the Cas13a-RNA complex by docking RNA onto Cas13a using a hybrid algorithm of template modeling and ab initio free docking; (8) optimize the 3D-structure of the Cas13a-RNA complex using GROMACS; (9) docking simulation of the target RNA and Cas13a using ZDock to predict the interaction pattern; (10) select the most stable complex for visualization and analysis using PyMol.The detailed steps are as follows: (1) predict the secondary structure of the crRNA using RNAfold and optimize using GROMACS; (2) search the PDB database for Cas13a-nucleic acid receptor complexes; (3) dock the crRNA on the Cas13a using a hybrid algorithm of template modeling and http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) software, which also yielded the secondary structures of input RNA sequences. The 3DRNA (http://biophy.hust.edu.cn/3dRNA) was used to generate 3D-RNA structures. In parallel, the bDNA tool (http://www.scfbio-iitd.res.in/software/drugdesign/bdna.jsp) was used to generate an RNA complementary fragment. The RNA sequence with the highest qualified structure was selected for visualization analysis using PyMol v1.60.The non-complementary regions of four crRNAs were predicted using the RNAfold . For the crRNA and Cas13a model generation, the PDB library was searched for Cas13a-nucleic acid receptor complexes (PDB ID: 5XWP). ZDock was used for the docking simulation. Using PyMol v1.60, the conformation with the lowest energy was chosen for visual analysis across all feasible spatial conformations and interaction patterns.IDH1http://bioinfolab.miamioh.edu/CRISPR-RT) and were in vitro synthesized by Integrated Biotech Solutions . Lipofectamine-3000 reagent  was used to transfect crRNAs (300 ng/mL), according to the manufacturer's specifications. The following reporter RNAs were produced by Abbots Biotech  for this study.CrRNAs were created using the CRISPR-RT design tool (crRNA-1 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGACCUAUGAUGAUAGGUUUUACCCAUCC-3'crRNA-2 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUGACCUAUGAUGAUAGGUUUUACCCAUC-3'crRNA-3 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAUGACCUAUGAUGAUAGGUUUUACCCAU-3'crRNA-4 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGAUGACCUAUGAUGAUAGGUUUUACCCA-3'crRNA-5 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUGAUGACCUAUGAUGAUAGGUUUUACCC-3'crRNA-6 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAUGAUGACCUAUGAUGAUAGGUUUUACC-3'crRNA-7 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCAUGAUGACCUAUGAUGAUAGGUUUUAC-3'crRNA-8 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCAUGAUGACCUAUGAUGAUAGGUUUUA-3'crRNA-9 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAGCAUGAUGACCUAUGAUGAUAGGUUUU-3'crRNA-10 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAAGCAUGAUGACCUAUGAUGAUAGGUUU-3'crRNA-11 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUAAGCAUGAUGACCUAUGAUGAUAGGUU-3'crRNA-12 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAUAAGCAUGAUGACCUAUGAUGAUAGGU-3'crRNA-13 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCAUAAGCAUGAUGACCUAUGAUGAUAGG-3'crRNA-14 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCCAUAAGCAUGAUGACCUAUGAUGAUAG-3'crRNA-15 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCCCAUAAGCAUGAUGACCUAUGAUGAUA-3'crRNA-16 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCCCCAUAAGCAUGAUGACCUAUGAUGAU-3'crRNA-17 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUCCCCAUAAGCAUGAUGACCUAUGAUGA-3'crRNA-18 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAUCCCCAUAAGCAUGAUGACCUAUGAUG-3'crRNA-19 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGAUCCCCAUAAGCAUGAUGACCUAUGAU-3'crRNA-20 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUGAUCCCCAUAAGCAUGAUGACCUAUGA-3'crRNA-21 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUUGAUCCCCAUAAGCAUGAUGACCUAUG-3'crRNA-22 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAUUGAUCCCCAUAAGCAUGAUGACCUAU-3'crRNA-23 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUAUUGAUCCCCAUAAGCAUGAUGACCUA-3'crRNA-24 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUAUUGAUCCCCAUAAGCAUGAUGACCU-3'crRNA-25 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUGUAUUGAUCCCCAUAAGCAUGAUGACC-3'crRNA-26 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCUGUAUUGAUCCCCAUAAGCAUGAUGAC-3'crRNA-27 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUCUGUAUUGAUCCCCAUAAGCAUGAUGA-3'crRNA-28 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCUCUGUAUUGAUCCCCAUAAGCAUGAUG-3'crRNA-29 5'-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCUCUGUAUUGAUCCCCAUAAGCAUGAU-3'Reporter RNA:5-nt: FAM-UUUUU-BHQ10-nt: FAM-UUUUUUUUUU-BHQ15-nt: FAM-UUUUUUUUUUUUUUU-BHQ20-nt: FAM-UUUUUUUUUUUUUUUUUUUU-BHQ5-107 cells) by instantly lysing cells on the petri dish. Chloroform (0.2 mL/1 mL of TRIzol) was then added and vigorously agitated for about 3 min before centrifuging at 12,000 g for 15 min at 4 °C. The upper aqueous layer was transferred to a fresh Eppendorf tube, mixed with 0.5 mL of isopropanol to precipitate RNA, and incubated at 4 °C for 10 min, before centrifuging at 12,000 g for 10 min. Pellets were spun for 5 min at 7,500 g at 4 °C in 75% ethanol, vacuum or airflow dried for about 10 min and dissolved in 15 µL of RNase-free water. The Prime Script RT Kit  was used to synthesize the cDNA, according to the manufacturer's instructions. The LightCycler2.0  was used for the qRT-PCR analysis in triplicates, and Ct values were normalized to that of the GAPDH as an internal control. Relative quantification values for target genes were obtained based on the 2-∆∆Ct method. Three biological replicates were carried out for each experiment. The following primer pairs were used in this assay:The TRIzol reagent (Invitrogen) was used to extract the total RNA from treated and untreated cells (0.5 mL per 1x10GAPDH-F: 5'-TGCACCACCAACTGCTTAGC-3'GAPDH-R: 5'-GGCATGGACTGTGGTCATGAG-3'Cas13a-F: 5'-TGGAAAAGTACCAGTCCGCC-3'Cas13a-R: 5'-TCGAAGTCCTCGGTCACTCT-3'IDH1mut-F: 5'-CCAACTGCACACACGACCT-3'IDH1mut-R: 5'-TCCTCCTGTGTCGCCTTTAC-3'HOTAIR-F: 5'-ATAGGCAAATGTCAGAGGGTT-3'HOTAIR-R: 5'-TCTTAAATTGGGCTGGGTC-3'Puromycin (4 g/mL for selection) was used to select IDH1mut positive cells, which were subsequently transfected with LvCas13a for at least 48 h before the experiment, according to the manufacturer's instructions. Reporter RNAs were transfected in U87-IDH1mut-Cas13a cells using Lipofectamine-3000 . U87- and TBD0220-Cas13a-IDH1mut-reporter RNA cells were transfected with crRNA-14, synthesized by Shanghai Integrated Biotech Solutions Co. Ltd. using Lipofectamine-3000 (Invitrogen). At 6 h post-crRNA14 transfection, cells were examined under a confocal microscope and analyzed by flow cytometry. The efficiency of the Cas13a-based reporter system was also examined using flow cytometry and confocal laser scanning microscopy (CLSM).Plasmid DNA (pCas13a and pCrRNA) and reporter RNA  were diluted in transfection buffer and mixed at various molar weight ratios by vortexing. After 10 min of incubation at room temperature, a 20 µL volume of the mixture was analyzed using 0.6 percent agarose gel electrophoresis .GBM cells were first grown on coverslips, followed by transduction with targeting crRNA and reporter RNA vectors; then cells were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature and permeabilized with 0.1% Triton X-100 for 15 min. Confocal images of cells were captured using a confocal microscope (FV500) and analyzed with FluoView software .Mice (n = 3) were sacrificed on day 21 post-implantation and transcardially perfused with cold 4% PFA in PBS. After the fixation step, brains were dehydrated by sequentially placing them in 20% and 30% sucrose solutions, then embedded in optimal cutting temperature (OCT) compound , frozen at -80°C, and coronal sections (8micron) were prepared. Following that, frozen sections were fixed with 4% PFA for 30 min before permeabilizing with 0.2% Triton X-100 in PBS. CLSM  was used to acquire images.. In vitro, a minimum of 5,000 events were collected for each sample. Cell capture was done immediately on a flow cytometer (BD FACSCanto II) using the FACSDiva software and analyzed with Flowjo 10.6.2.The flow cytometry experiment was carried out in the same manner as described elsewhere. U87 and TBD0220 cells were treated, then digested with 0.25% trypsin and fixed with 4% PFA(1) 100 mg of PEI-NH2 and 150mg of BLG-NCA were separately dissolved in 2 mL dry N,N-Dimethylformamide (DMF) by heating and stirring in the N2 environment, and were allowed to react at 30° for 72 h. After the reaction, the product was added dropwise to a large amount of ice-cold ether, centrifuged, and dried in a vacuum chamber to obtain the PEI-PBLG. (2) 100 mg of PEI-PBLG was dissolved in 2 mL DMSO, then added with 25 mg of NHS-PEG5K-NHS, and allowed to react at room temperature for 30 min. After that, the cRGDfk peptide (1.1 eq.) was dissolved in triethylamine (3.0 eq.) and added to the previous reaction mixture to react at room temperature for 12 h; then the reaction solution was transferred to a dialysis bag (MW 1000 Da), dialyzed in pure water for 24 h, collected the dialysis solution and freeze-dried the product.5 cells per well and cultured for one day before co-culturing with nanoparticles. TOTO-3-labeled cRGD-PP (1 μg pDNA) was then added to the cells and incubated in a complete culture medium for 2 h. Following the incubation, the cells were rinsed with ice-cold PBS and fixed with 4% PFA at room temperature for 15 min. Subsequently, the cell nuclei were counterstained with DAPI and F-actin filaments with rhodamine-phalloidin, following the manufacturer's protocols. Then, images were captured under a CLSM .CLSM was employed to investigate the cellular uptake efficiency of cRGD -PEI-PBLG. U87 cells were seeded in a 35-mm confocal dish (Ф = 15 mm) at a density of 1×10mut or F3-T3 overexpression constructs before being injected into mice. On day 7, homozygous mice were randomly divided into two groups and given intravenous (i.v) injections of relevant formulations and prepared both sham and treated mice were for fluorescence imaging for the real-time detection of the reporter RNA transfer in vivo. The injection formation contained cRGD-PP-nucleic acid complex at a 1:1 (w/w) ratio. All interventions contained 30 µg of nucleic acid per dose, and the nucleic acid composition was prepared by mixing pCas13a, pcrRNA, and reporter RNA at a 1:1:4 (m/m/m) ratio.Tumor tissue harvested from the PDX model was processed into a single-cell suspension. The cells were infected with either IDH1Tumor tissue from the PDX model was separated into a single-cell solution. These cells were then cultivated with either the IDH1mut or F3-T3 overexpression constructs, and subsequently injected into the animals. Tumor growth was measured with bioluminescence imaging on days 7, 14, and 21. Intravenous injections contained cRGD-PP/pDNA mixture at a 1:1 (w/w) ratio. All interventions contained 10µg of pDNA per dose, where pDNA denoted as pCas13a/pcrRNA = 1:1 (m/m). The solutions were administered by tail vein injection every other day for 21 days. On day 21, mice were sacrificed, and brain tumors were harvested and processed for Hematoxylin and Eosin (H&E) staining and immunohistochemical (IHC) analysis. In parallel, another set of animals was utilized for the Kaplan-Meier survival curve analysis.Tissue sections of 7 µm thickness were cut from the formalin-fixed paraffin-embedded (FFPE) tissue blocks, then de-paraffinized and hydrated in deionized water. After that, antigen retrieval was performed in the citrate buffer (pH 6.0) for 30 min at 95 °C. Sections were treated for 12 h at 4 °C using anti-Ki67 antibody at 1:1000 dilution , followed by incubation with a respective secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. For visualization, a DAB substrate solution was applied. Digital images were captured using a brightfield microscope.t-test or one-way analysis of variance (ANOVA). For statistical analysis and data plotting, GraphPad Prism 8.0 was used. PyMol v1.60 was used for visualization and analysis of the lowest energy conformation. Data are expressed as mean ± standard error (S.E.). ns indicates no statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.For more than two groups, all data were compared using an unpaired R132H point mutation using a tiling array of 29 RNA sequences and chose the top-scoring crRNA from a set of four using in silico analysis . Then, we conducted a detailed bioinformatics analysis to establish a Cas13a-based strategy that could selectively target and cleave the IDH1 mutant mRNA . Binding energy calculations were performed to assess the relative propensity of interactions between different crRNAs, the target RNA, and Cas13a. The docking scores were also calculated for individual single-stranded (ss) crRNA to determine the binding affinity of the Cas13a in each interaction, which ranged from -2023.14 to -1685.98kJ/mol. Notably, the Cas13a-crRNA binary complex exhibited an average docking score of -1844.74kJ/mol . Besides, among the twenty-nine potential crRNA sequences, the docking scores of binary interactions for crRNAs #10, 14, and 22 were in the lowest range (SupplementaryTable S1). Following the crRNA binding to its respective target RNA via sequence complementarity, the Cas13a gets recruited to the target site and mediates the splicing of the target RNA. Based on the standard Cas13a-RNA complex template, different combinations of Cas13a-RNA binary complexes were constructed from scratch utilizing a hybrid template modeling and the free-docking approach (PDB ID: 5XWP). Docking scores between the crRNA-target RNA duplex and Cas13a were low, ranging from -2533.62 to -2023.00kJ/mol. While the median docking score of the ternary complex was -2354.48kJ/mol . Notably, the docking scores for crRNAs 10, 14, and 22 bound to the Cas13a were in the lowest range among 29 crRNAs, indicating that these crRNAs had the highest potential in forming the Cas13a-guided binary complexes (SupplementaryTable S2). The docking score was negatively correlated with the interaction strength and stability of the composite system. As shown in Figures , docking scores of 10, 14, and 22 for crRNAs bound to the Cas13a were similar and in the lowest range among 29 crRNAs, indicating that these crRNAs had the highest potential to form Cas13a-guided binary complexes. This process narrowed down our screening for crRNAs. In silico studies on the 29 crRNA candidates yielded the best crRNA structure with the following properties: crRNA-14 contained a 28-nt long guide region , including a 38-nt protein binding region (G(-38)-C (-1), in orange), which was used for modeling . The 3D structure of the crRNA-14 was optimized and visualized using the GROMACS 4.6 dynamic program . The non-complementary portion (protein-binding region) of the crRNA with a hairpin structure qualified for the stabilization of the crRNA/Cas13a/target RNA ternary complex. The complementary sequence (guide region) of the crRNA for the target RNA can be identified via scanning for the most suitable complementary sequence, which then directs the Cas13a to cleave the target RNA at its recognition sequence. These two properties, when combined, guarantee an accurate RNA cleavage by Cas13a mut mRNA sequence were then combined to analyze the probability of forming a 28-bp guide-target RNA complex that resembled an A-form helix . Further examinations of the electrostatic binding forces on the surface of the Cas13a-crRNA14 complex revealed that the Cas13a's catalytically active residues were positively charged, and any of the exposed oxygen atoms of crRNA-14 could also act as the positively charged sites in the Cas13a complex, promoting the binding of the crRNA14 to the IDH1mut mRNA, thereby enhancing the target RNA cleavage effectively .In glioma patients, missense mutations in IDH1 protein, including the most commonly occurring R132H mutation, are prevalent R132H mRNA, showing that the crRNA14-IDH1R132H RNA duplex had a strong binding affinity for the Cas13a's catalytic groove, where key residues, such as Met-1, Thr-4, Lys-5, Arg-41, Val-553, Ser-555, Tyr-601, and Lys-1124, created strong hydrogen bonds (H-bonds) with bases in the 16-29-nt region of the crRNA-14 . Furthermore, Lys-855, Thr-557, Asp-590, Asn-804, Arg-809, and Lys894 were also found to form strong H-bonds with nucleotides in the 51-65-nt segment of the crRNA-14, improving the crRNA-14's binding affinity with the IDH1R132H RNA, and ensuring effective cleavage of the target sequence . The ability of the crRNA to form multiple H-bonds with its target RNA sequence was found to be crucial for the formation of energetically stable Cas13a ternary complex and crRNA-guided cleavage of the target RNA sequence. Collectively, these results suggest a theoretical basis for the Cas13a-mediated RNA cleavage at the molecular level.Next, we carried out an in-depth analysis of interactions between the Cas13a, crRNA14, and IDH1Figure ). To test the cleavage efficiency of the crRNA, in vitro knockdown assays were performed in cells over-expressing the IDH1mut variant. The IDH1mut mRNA level was then determined by qRT-PCR assay, taking GAPDH as the internal control. The knockdown efficiencies of crRNAs-14 and -22 were in the highest range . Together with the binding energy scoring, these findings demonstrated that crRNA14 and crRNA22 had the strongest knockdown efficiency, which was in agreement with in silico predicted results. Hence, we randomly chose the crRNA14 for subsequent experiments. Sequence analysis of IDH1 in 939 tumor samples revealed 161 somatic mutations at residue R132, including R132H (142 tumors), R132C (7 tumors), R132S (4 tumors), R132L (7 tumors), and R132G (1 tumor). To investigate the specificity of this strategy, we performed a knockdown assay by transfecting crRNA-14 in U87 and TBD0220 cell lines over-expressing five different target genes. The qRT-PCR results showed that crRNA-14 exhibited the strongest knockdown only in R132H over-expressing cell lines . Total RNA integrity was significantly reduced in U87-IDH1mut-Cas13a cells at 24 h post crRNA-14 transfection , while it remained unaffected in U87-Cas13a cells , as measured by the Agilent Bioanalyzer. In U87-IDH1mut-Cas13a-crRNA-14 cells, the RNA integrity number (RIN) and the ratio of 28S to 18S were simultaneously reduced . Likewise, experiments on the TBD0220 cell line with the Cas13a-based IDH1mut knockdown demonstrated comparable findings . Furthermore, the Cas13a mRNA expression was significantly decreased in the IDH1mut-positive groups . The expression of HOX Transcript Antisense RNA (HOTAIR) RNA, a random and intrinsically expressed RNA, was also equally decreased by 50% in both Cas13a and IDH1mut-expressing cell lines transfected with crRNA-14 . These data indicated that the Cas13a system had no obvious off-target effect and might have induced collateral effects after targeting the IDH1mut mRNA. In this context, it can be suggested that crRNA-14 may elicit efficient and stable cleavage activity of the target RNA and trigger collateral cleavage.First, we confirmed by DNA sequencing that none of the U87 and TBD0220 cell lines had any pathogenic IDH1 mutation . First, we employed flow cytometry to detect fluorescence changes in each group at 6 h post-crRNA-14 transfection in U87- and TBD0220-Cas13a-IDH1mut-reporter RNA cells, showing that 5-nt and 10-nt long reporter RNAs had negligible detection ability, while the 20-nt reporter exhibited a significant change in the fluorescence level as compared to that of the positive control . Consistently, CLSM was employed to confirm these comparable findings for the 20-nt long reporter RNA group with respect to the positive control . These data demonstrated that the 20-nt length of the reporter was optimum for subsequent investigations. Under the controlled condition, the quencher-tagged reporter RNAs did not fluoresce unless the activated Cas13a recognized its RNA target and cleaved both on-target and adjacent off-target RNAs, including the reporter RNA, resulting in the emergence of the fluorescence signal . To further demonstrate the efficacy of the system, we determined the proportion of IDH1mut expressing U87 and TBD0220 cells with increased fluorescence intensities than that in IDH1mut-negative control cells by flow cytometry . Moreover, confocal imaging also indicated that only the IDH1mut-expressing cells had fluorescence signals, while no fluorescence was detected in IDH1mut-negative control cells (Supplementary). Previously, we reported that the FGFR3-TACC3 (F3-T3) fusion gene chimera might be utilized to treat glioma, which highlighted the more or less exact function of the Cas13a-based tool in glioma therapy Supplementary). Besides, we used the Cy5.5-labeled reporter RNA for bioluminescence imaging that showed only IDH1mut expressing cells produced fluorescent signals . Taken together, these data suggest that the optimized reporter RNA system could be highly specific for in vitro molecular detection of the target RNA degradation products, thus confirming an efficient cleavage by the Cas13a-crRNA system.To further investigate the collateral effect of the Cas13a system, we attempted to detect specific nucleic acid fragments which were generated by Cas13a cleavage through the introduction of fluorescent-tagged reporter RNAs. Four reporter RNAs of different lengths were designed for the flow cytometric and immunofluorescence (IF) analyses to screen and determine the optimal reporter RNA sequence for subsequent experiments . To improve the delivery efficiency of the Cas13a-crRNA complex, a cRGD peptide-conjugated PEI-PBLG (PP) Figure ). DLS and TEM measurements revealed the formation of spherical nanoparticles with an average diameter of ~100nm , which was considered effective for successful endocytosis. These complex particles had a zeta potential of -0.10 ± 1.04 mV. To assess the effectiveness of cellular uptake, TOTO-3-labeled pDNA (green) was incorporated into the polyplex formulation and incubated with U87 and TBD0220 cells for CLSM. Significantly high fluorescence intensities were observed in cRGD-PP-treated cells, demonstrating that the cRGD-PP may successfully transfer pDNA into cells  To better exploit this unique Cas13a-crRNA editing system, we aimed to design an integrated platform for the diagnosis and treatment of diseases like glioma, requiring efficient and precise genetic manipulations. As mentioned before, we established a reporter RNA (quenched fluorescent RNA)-based strategy to create a Cas13a-based platform for integrating the diagnosis and treatment of glioma under traceable conditions . According to the ex vivo fluorescence images, mice injected with cRGD-PP(Cas13a/crRNA) exhibited strong fluorescence in the brain, indicating effective BBB penetration . Nanocapsule distribution and organ accumulation were assessed by ex vivo organ luminescence in patient-derived xenograft GBM mouse model followed by intravenously injecting nanocapsules via tail vein. Stronger fluorescence was observed in the brains of mice treated with cRGD-PP(Cas13a/crRNA) compared to the free Cas13a/crRNA controls .To further evaluate the ability of the copolymer complexes to cross the BBB in vivo, we used an orthotopic TBD0220-Luc GBM mouse model. TOTO-3-labelled nanoparticles were injected via the tail vein and, after administration, the signal from the complexes was monitored using an IVIS. The images showed that the fluorescence signal from the brain began to appear at 1 hour post-injection and its intensity was maintained up to 24 hours  injection in animals . Our previous work was the first to demonstrate that the mutant F3-T3 gene chimera could be used as a new therapeutic for the treatment of glioma and thus clarified the precise role of the CRISPR/Cas13a system in tumor treatment mut mRNA. Bioluminescence imaging results revealed that in an intracranial GBM mouse model containing the IDH1 mutation, i.v injected mice with crRNA14 exhibited a significant shift in their fluorescent signal intensities compared to the sham controls similarly i.v injected with the crF3-T3 . According to ex vivo fluorescence images, crRNA14-injected mice had increased fluorescence signal intensities compared to that of the control animals injected with the crF3-T3 via the tail vein . The PDX model without IDH1 mut constructs incubation has been used as a control to prove the sensitivity and accuracy of this detection system in vivo diagnosis. The results revealed that in an intracranial GBM mouse model without the IDH1 mutation, i.v injected mice with crRNA14 or crF3-T3 did not exhibit a shift in their fluorescent signal intensities (Supplementary). According to ex vivo fluorescence images, neither crRNA14-injected mice nor crF3-T3-injected mice had increased fluorescence signal intensities (Supplementary). Simultaneously, in another intracranial GBM mouse model harboring the F3-T3 fusion gene, crF3-T3-treated mice showed significantly high fluorescent signals compared to that of control animals injected with the crRNA14 . Notably, the results obtained from the analysis of ex vivo bioluminescence images were consistent with those obtained from in vivo bioluminescence images .To perform Figure ), thus eliminating the possibility of a non-specific fluorescence signal of the reporter. In summary, we explored a molecular detection platform based on the Cas13a RNA-editing system, where the selective designing of an efficient crRNA and a corresponding fluorescent-quenched reporter RNA would enable us to monitor and visualize the targeting efficiency of the Cas13a system at a molecular level.Confocal microscope images of the GBM mice brain sections indicated that the Cy5.5 signal could only be detected in brain tissues expressing the target gene . By day 7, both cell types exhibited tumorigenesis. In comparison to IDH1mut-overexpressing tumors treated with crF3-T3, IDH1mut-Cas13a-crRNA-14 cell-derived tumors showed a relatively slower growth rate over 21 days . The IDH1mut-Cas13a-crRNA-14 tumor-bearing mice had higher overall survival (OS) rates than those in the IDH1mut tumor-bearing mice treated with crF3-T3, according to the Kaplan-Meier survival curve analysis . In GBM mice, the Cas13a-crF3-T3 therapy resulted in a significantly decreased progression of the F3-T3-overexpressing tumor compared to that of the crRNA-14 control group . The Cas13a-crF3-T3-treated tumor-bearing mice had higher OS rates than those of the crRNA-14-treated control animals, as shown by the Kaplan-Meier survival curves . The reduction in the size of the Cas13a-crRNA-14-treated tumor, compared to that of the Cas13a-crF3-T3-treated tumor, was confirmed by H&E staining of tumor tissue sections . Additionally, the Ki67 staining in crRNA-14-treated IDH1mut-overexpressing tumors demonstrated a significant growth reduction . Similarly, administration of the crF3-T3 in mice overexpressing the F3-T3 gene chimera presented significantly reduced tumor volumes , which was consistent with the Ki67 staining in corresponding tissue sections . Cumulatively, these findings suggest that the Cas13a-based therapy can be a promising treatment option in effectively retarding tumor growth in GBM patients.In an intracranial GBM model of BALB/c nude mice, the Cas13a-based RNA -editing tool was tested for its potential to suppress tumor growth in both TBD0220-IDH1in vivo gene-editing could be achieved using lipid nanoparticles encapsulated CRISPR/Cas9 complex in vivo molecular diagnostics, especially in brain tumors.The CRISPR/Cas13a system has the advantage of being a fast, precise, highly sensitive, and programmable RNA-editing tool for molecular diagnostics mut RNA in a stacked tile and predicted the binding stability between the Cas13a, crRNA, and corresponding target RNAs based on the computer simulation methods. Molecular docking techniques allowed us to model the binding interaction between the crRNA and the target RNA. Using structural information and computational algorithms, molecular docking can predict the potential binding positions and affinity of a particular crRNA for its target RNA. By screening a wide range of target sequences, the optimal crRNA-target RNA pair with the most favorable binding interactions and the highest enzymatic efficiency can be identified. Overall, these computational approaches facilitate the identification of the most effective Cas13a-mediated RNA cleavage system, thereby improving the precision and efficiency of the genome editing and diagnostic application of the CRISPR-Cas13a system. More importantly, it has the potential to improve screening efficiency and minimize off-target effects.Herein, we designed 29 crRNAs targeting the IDH1mut mRNA for subsequent experiments. To save time and avoid evaluating a good deal of possible crRNA sequences, we used a variety of bioinformatic tools to narrow down the list of crRNAs that had significantly higher binding affinities for the target RNA sequence. Afterward, we designed four reporter RNAs of different lengths to use them for flow cytometry and IF experiments in screening for the optimal reporter RNA length, which was found to be 20-nt in this study. To track down the target RNA degradation in live-cell imaging, we performed an end-labeling reaction to generate Cy5.5-labelled reporter RNA for bioluminescence imaging. To improve the delivery efficiency of the Cas13a/crRNA complex in GBM mice, we coated plasmids expressing Cas13a and crRNA, as well as the reporter RNA with the cRGD-PP for i.v administration via tail vein. The results showed that the GBM mice harboring the IDH1 gene mutation and treated with crRNA-14 had significantly higher fluorescent signal intensities compared to that of the control mice injected with crF3-T3. We further recapitulated this experiment in GBM mice carrying the F3-T3 gene mutation to revalidate the results. These results, therefore, highlight the therapeutic potential of the Cas13a/crRNA technique for GBM and any related tumor treatments. Binding of the target RNA by the Cas13a/CRISPR RNA (crRNA) complex activates the RNase activity of Cas13a, leading to non-specific cleavage of cellular RNAs and eventually inducing programmed cell death or dormancy in vivo imaging method to detect the target RNA in glioma cells/tissues, suggesting the possibility of a tailored therapeutic approach for the diagnosis and treatment of difficult-to-treat cancers such as gliomas.Computed structural simulations play a crucial role in narrowing down the screening range of potential crRNAs. While this method serves as a valuable tool guiding our decision-making process, it should not be considered an infallible substitute for real-world experiments, but rather as a complementary approach to increase the screening efficiency and targeting precision in experimental models. Combined with the qRT-PCR-based quantitative screening of the top-scoring crRNAs, we finally selected the crRNA-14, offering the most effective cleavage of the IDH1in vivoAchieving an effective and detectable level of Cas13a/crRNA components inside the target cell remains a huge challenge, especially in hard-to-transfect cell types. One strategy to address this problem is to use smaller homologs of Cas13a or minimization of the system for their efficient loading into virus particles. While another approach could be the utilization of specific types of nanomaterials that can facilitate the membrane penetration by the Cas13a/crRNA complex in GBM cells. In a recent study, we have described a multistage delivery method of nanoparticles that can cross a wide range of physiological barriers and effectively supports the CRISPR/dCas9-mediated tumor growth inhibition Supplementary figures and tables.Click here for additional data file.


Fetal haemoglobin (HbF) remains a major sickle cell disease modifier. The mechanism of HbF synthesis has been studied for several decades with the intention of increasing interventions for sickle cell disease (SCD), including drugs. However, the complex mechanism of HbF synthesis is influenced by multiple genetic factors interacting with environmental factors. In order to capture useful genetic information, especially with limited resources, one has to carefully design the study. This includes choosing the relevant participants, the correct phenotyping, the choice of samples, and the right genomic assays. This paper describes the approach undertaken as part of preparations for a reticulocyte transcriptome study intended to discover genes associated with HbF decline in newborns in Tanzania.Of the 152 newborns enrolled in the larger study, 40 babies were selected for the reticulocyte transcriptome study based on their HbF levels at birth and later stage of life. Of these, 30 individuals were included under the category of high HbF levels ranging from 72.6–90% and the remaining 10 under the category of low HbF levels ranging from 5.9 – 10.3%. The reticulocyte enrichment recovery purity ranged from 85% - 97%. The total RNA concentrations obtained were >250 ng total RNA, with the average purity of 1.9 (A 260/280) respectively. The total concentration obtained was sufficient for the transcriptome and other downstream assays.We have documented important steps and factors to consider in identifying the relevant participants and required laboratory sample processes prior to the final stage, which involves total reticulocyte RNA sequencing. Interventions for lifetime conditions such as sickle cell disease (SCD) will be more beneficial if tailored in a personalized manner. Fetal haemoglobin (HbF) is a known major SCD modifier and therefore a target for interventional developments, including drugs –3. The nUnderstanding the differential gene expression during HbF decline is expected to contribute to the search for potential drug targets as well as gene therapy for SCD. One of the approaches has been to investigate genes that are switched on and off in different cell populations at different stages of erythropoiesis , 9. SincIn order for such a study to generate relevant data and due to cost limitations for such genomic studies, it is important to conduct a careful selection of participants, preferably those with extreme HbF levels. Studying individuals with extreme phenotypes (EP) enhances the identification of genetic variants that are strongly associated with a particular trait or disease, in this case HbF. Such varA total of 152 newborns were screened for the reticulocyte transcriptome study from December 2019 to November 2022. A total of 40 individuals were found to fit under the extreme category, 30 with extreme high HbF  and 10 with extreme low HbF  .A recovery purity of >85% was achieved following the adopted enrichment assay. This purity was mainly achieved through leukofiltration by Ficoll and Percoll centrifugation media coupled with negative selection by CD45+. A flow cytometry analysis was used to verify the purity .Recovered concentration of RNA upon measurement by qubit fluorimeter 3.0 was >250 ng total RNA for all the samples and the purity of RNA as measured by Nanodrop at the absorbance ratio of 260/280 was average of 1.96.Identifying genetic factors associated with fetal haemoglobin remains an important gateway to increasing alternative interventions for the management and treatment of SCD. However, identifying the right genetic factors requires a robust design, taking into consideration many factors that are not always straightforward. This is more difficult in low and middle - income countries where resources are limited. This paper describes the approach undertaken in Tanzania as part of an ongoing study aimed at studying the reticulocyte transcriptome in newborns with high and low levels of fetal haemoglobin. We focused our transcriptome study on selected newborns at birth and at a later stage, which provided us with those with extreme HbF levels (very high/low HbF). This approach is scientifically backed up and proven to enhance the identification of genetic variants, study disease mechanisms, enhance statistical power, contribute to personalized medicine, and study genetic architecture.The selection of participants was based on the distribution of HbF levels established in our population. The individuals included in the extreme high HbF category were those above the upper quartile. Our study design, which involved measuring HbF at birth (using cord blood), provided us with a good selection of individuals with very high HbF levels (72.6–90.2). This is true because HbF is at the highest level at this point in any individual’s life. For the participants with low HbF (5.9–10.3) we selected babies at 18 months, at which HbF has declined to a significant level. Our original plan was to include both babies with very high and low HbF at birth. However, it was challenging as most babies had very high levels of HbF. Studies with a larger sample size that can allow for this design may benefit from studying and comparing the gene expression profiles of babies with extreme HbF levels at birth.Our approach prioritized studying reticulocyte transcriptomes over other erythroid cell populations. This is because they are a cell population synthesized at the final stages of erythroid differentiation. Reticulocytes are also the only cell population with remnant genetic material with robust descriptions of genes, including those being highly expressed. However, reticulocytes are just a small proportion of cells found in either the cord (3.75 ± 1.16% with a range of 1.41–8.4%) or peripheral blood (2–6%). Therefore, in order to get sufficient cells for the transcriptome, the reticulocytes should be enriched and purified. We adopted the reticulocyte enrichment method by Skulski et al., 2019 . EnrichmConcentrations of RNA recovered from both high and low categories ranged from 280–1500 ng total RNA, which is sufficient for the transcriptome study. Due to the sensitivity of the RNA sequencing assay it was necessary to ensure that the isolated RNA was of good quality. This was achieved using analysis by Qubit and through RNA integrity check using gel electrophoresis. On average RNA purity for our samples was sufficient for the transcriptome study, however better quality could be achieved by utilising more efficient RNA isolation systems and increasing the starting number of reticulocytes. Similarly, RNA integrity and intact assessment by gel electrophoresis indicated a non- degraded RNA with no contamination of gDNA as shown in Fig. 4We have provided a detailed account of the targeted approach for implementing a reticulocyte transcriptome study in our population. Our expectations are, this study will contribute significantly to the genomics of HbF especially in identifying potential targets for interventions which have a great impact for individuals with SCD.The study was conducted at Mbagala and Sinza hospitals after obtaining ethical clearance from the Muhimbili University Institutional Ethics Review Board (IRB). The study involved newborns and infants (below the age of 18 months). Informed consent was obtained prior to enrolment into the study.Cord blood collection was conducted by well trained nurses following the standard cord blood collection protocol. ImmediaFetal haemoglobin quantification was determined by using variant high performance liquid chromatography (VNBS-HPLC) . Briefly, samples were mixed properly in a Stuart rollermixer SRT6D for 5 minutes at a speed of 35 revolutions per minute (RPM). In a 96 well plate, 5ul of blood was added in a well followed with 250 ul of distilled water and eluted for 30 minutes in a Heidolph Titramax shaker, which was then run in a VNBS Biorad HPLC machine . The extLeukofiltration, Washing of beads, Negative selection with CD45 + cells, and Enrichment of CD71 + cells as adopted from previously reported methods  from Thermo Scientific U.S.A. A total of five  steps we™ RNA High Sensitivity (HS) assay kit. Prior to every run, the Qubit 3.0 fluorometer was calibrated using the standards provided in the kit, and each run control was used.After extraction, RNA was purified with a (1:1) RNA-DNase ratio and incubated for 15 minutes at 37°C, thereafter passed in a spin column to remove any gDNA contamination. Two instruments were used for determining the quantity and purity of isolated RNA, namely the Qubit Fluorometer 3 and NanoDrop spectrophotometer, respectively. During measurement by Nanodrop, RNase free water was used as the blank solution before each sample measurement. 2μL of RNA were placed on the pedestal, the lid was closed, and the RNA concentration was measured. Measurement by Qubit Fluorometer 3.0 was done using the Qubit™ XR + with Image was used for visualization using lab software from Bio-Rad Laboratories Inc, USA (The gel electrophoresis method was used to check RNA integrity. RNA with a known concentration was run through a gel electrophoresis in order to visualize RNA intactness, size, and overall integrity. Agarose gel of 0.8% was used for the integrity analysis. Molecular Imager Gel DocInc, USA . Upon wa


Hepatitis A diagnosis relies on serology and occasionally on hepatitis A virus (HAV) RNA detection. For timely diagnosis and the avoidance of drawing additional blood, molecular testing is often performed as reflex testing by using blood specimens that were initially sent for anti-HAV serology. Reflex molecular testing is preferably performed from different sample aliquots, but, for limited sample quantities, it uses samples that have been preprocessed in an immunoassay analyzer. In 2012, we first observed sporadic HAV RNA-positive cases that were inconsistent with patients’ serological profiles and/or medical histories, suggesting that occasional laboratory contamination was causing false-positive PCR results. Multiple external quality assurance (EQA) and laboratory surface contamination checks were performed, questionable specimens were tested with various HAV RNA tests, and follow-up serum/stool samples were collected. All contamination-check samples and samples from healthy individuals tested HAV RNA-negative, and the laboratory successfully passed all EQAs. The HAV RNA-positive results were reproducible with various HAV RNA assays. No patients seroconverted, and their follow-up samples were consistently HAV RNA-negative. Finally, a detailed review of testing protocols revealed a correlation between HAV RNA false positivity and preceding anti-HAV testing with the Cobas-e411 automated immunoassay analyzer. HAV RNA was detected in the Cobas-e411 anti-HAV reagents, with the HAV sequences matching those from the false-positive samples. Preceding anti-HAV testing using two other immunoassay analyzers did not result in subsequent HAV RNA false positivity during reflex testing. The Cobas-e411 pipetting procedure with a single pipette tip collecting samples and anti-HAV reagents contaminated the original sample with the HAV RNA that was present in the immunoassay’s reagents, thereby resulting in HAV RNA false positivity during the reflex testing.IMPORTANCE We present the first report of sporadic HAV PCR false-positive results that have been observed during the reflex testing of serum samples that have previously been tested for anti-HAV antibodies and have been caused by contamination with HAV RNA that is present in the reagents of the commercial anti-HAV immunoassay, with potentially serious clinical consequences. Although HAV RNA was consistently detected in the anti-HAV reagents of all three automated immunoassay analyzers that were in use in our laboratory, only the use of one analyzer and the corresponding commercial anti-HAV immunoassay reagents resulted in contamination that led to false positive HAV RNA results, and this was due to a peculiar pipetting mode of action in which the analyzer uses a single pipette tip to collect both anti-HAV reagents and a sample, which consequently causes the permanent contamination of the original sample with HAV RNA. Manufacturers should strongly consider the occasional need for reflex molecular testing from preprocessed samples and design their analyzers in a way that prevents contamination. Acute hepatitis A has a relatively long incubation period (15 to 50 days). Fecal-oral transmission caused by contaminated drinking water, poor sanitation, and personal hygiene is by far the most frequent transmission route, and this is followed by oral-anal contact, as accounted only recently (https://www.ecdc.europa.eu/en/hepatitis-A/facts). Although anti-HAV IgM testing is quite reliable, several false-positive anti-HAV IgM results have been reported, thereby highlighting the need for the cautious interpretation of positive results, always in the context of patient history and other laboratory and clinical parameters, and especially in low-incidence settings  , 10. ForTo the best of our knowledge, we present the first known report of HAV RNA PCR false-positive results observed during reflex HAV RNA testing and caused by the contamination of original blood samples with HAV RNA present in the reagents of the commercial anti-HAV immunoassay. The reason for occasional HAV RNA PCR false-positive results remained unresolved for 9 years, until it was linked with the preceding testing of serum samples for anti-HAV antibodies by one of the three automated immunoassay analyzers that was used in the laboratory.Our viral hepatitis reference laboratory started routine HAV RNA testing in 2005. Unlike frequent requests for anti-HAV serology, HAV RNA testing was requested about 18 times annually , except for 2022, when, due to a HAV outbreak in Hungary, requests increased to a few hundred per year.In 2012, our laboratory started observing sporadic HAV RNA-positive cases that were inconsistent with patients’ anti-HAV serological profiles and/or medical histories, suggesting that these cases could be associated with occasional laboratory contamination. Several measures, described in detail in Materials and Methods, were taken to identify the potential source of HAV RNA contamination that was leading to the sporadic HAV RNA false-positive results. All serum samples with suspicious initial false-positive HAV RNA results repeatedly tested HAV RNA-positive using three commercial PCR assays as well as an “in-house” HAV RNA PCR test, both in our laboratory and in other laboratories at our institution, including laboratories that had never dealt with hepatitis A positive samples. The Sanger sequencing of the PCR products clearly confirmed the presence of HAV RNA in all of the samples that had a suspicious initial false-positive HAV RNA result, from which HAV sequences were successfully generated.However, the close follow-up of individuals that were initially anti-HAV IgM-negative (and HAV RNA “positive”) revealed that none seroconverted for anti-HAV antibodies in the following months. Similarly, all available follow-up serum and/or stool samples from individuals with suspected initial false-positive HAV RNA results clearly tested HAV RNA-negative. In addition, all of the PCR-contamination check samples that were collected from various laboratory surfaces, previously HAV-negative samples, blank water samples that were included in each PCR run, and serum samples that were obtained from nine healthy HAV RNA-negative individuals consistently tested HAV RNA PCR-negative.In addition, the laboratory was 100% proficient in all HAV RNA EQA control panels purchased, and no single HAV RNA false-positive result was detected when testing the HAV RNA-negative EQA panel samples.Finally, close inspection and a detailed step-by-step review of all of the testing protocols that were used in our laboratory revealed a correlation between the HAV RNA false positivity and the preceding testing of serum samples with Elecsys Anti-HAV or Elecsys Anti-HAV IgM assays on a Cobas e411 automated immunoassay analyzer , which is one of the three immunoassay analyzers that was used in our laboratory from 2012 to 2018. The preceding testing of serum samples for anti-HAV IgG and/or anti-HAV IgM antibodies with two other automated immunoassay analyzers that were in use, namely, a Vitros 3600  and an ARCHITECT i2000SR , did not result in subsequent HAV RNA false positivity during reflex HAV RNA testing. Therefore, the HAV RNA contamination of samples during the preceding testing for anti-HAV antibodies on the Cobas e411 was strongly suspected as a source of the sporadic HAV RNA PCR false-positive results that were recorded in our laboratory during the nine-year period.To confirm our hypothesis, we first tested four serum samples from four healthy individuals for the presence of HAV RNA, and all four tested HAV RNA-negative. Then, the same four samples were first tested on a Cobas e411 for the presence of anti-HAV antibodies using Elecsys Anti-HAV and Elecsys Anti-HAV IgM assays, and this was followed by reflex HAV RNA testing. All four samples that had been previously processed on a Cobas e411 clearly tested HAV RNA-positive, with the cycle threshold (CT) values ranging from 27 to 29. The presence of HAV RNA was also consistently detected in different batches of reagents of both the Cobas e411 Elecsys Anti-HAV and Anti-HAV IgM assays, with the CT values ranging from 20 to 24. HAV sequences were obtained from all four of the Cobas e411 anti-HAV preprocessed samples as well as directly from the Cobas e411 anti-HAV reagents, and they showed 100% identity. Furthermore, a phylogenetic analysis showed that these sequences were significantly different from all of the HAV sequences that were generated from patients with clearly confirmed HAV infections in Slovenia between 2012 and 2020 .Interestingly, although the presence of HAV RNA was also consistently detected in different batches of anti-HAV reagents from the two other automated immunoassay analyzers that were used in our laboratory (Vitros 3600 and ARCHITECT), due to the different pipetting modes of these two analyzers in comparison to the Cobas e411, HAV RNA false positivity was never detected in the HAV RNA reflex testing of samples that were preprocessed in either the Vitros 3600 or the ARCHITECT.Reflex molecular testing following serological screening/testing is a frequent and beneficial practice in virological laboratories that offer HIV and hepatitis testing services. Preferably, reflex molecular testing should be performed on a different aliquot of a sample than the one that was used for (preceding) serological testing or from another sample that has been concomitantly received from the same patient. Such a protocol has been a standard practice for years for reflex testing in our laboratory for HIV and hepatitis B and C, and, in recent years, also for hepatitis A, D, and E. However, when an unpredicted positive or intermediate serological test result is obtained and reflex molecular testing is not initially considered, as well as in the case of a limited quantity of available specimen, to ensure timely diagnosis and to avoid an additional blood draw, reflex molecular testing is also performed on samples that have been preprocessed in immunoassay analyzers. In most cases, such a practice is absolutely valid and beneficial, but, exceptionally, as described here, it could be risky and could have significant clinical consequences.To the best of our knowledge, this is the first report of HAV RNA false-positive results that have been detected during reflex HAV RNA testing and caused by HAV RNA that was present in the reagents of a commercial anti-HAV immunoassay. HAV RNA was consistently detected in different batches of the anti-HAV reagents of all of the three automated immunoassay analyzers that were in use in our laboratory , most likely due to the presence of the lysates of the inactivated (whole) viruses  that were used as antigens in the anti-HAV reagents instead of recombinant HAV antigens . However, only the use of one analyzer and the corresponding commercial anti-HAV immunoassay reagents resulted in contamination that led to false positive HAV RNA results. The principal reason for the false positive HAV RNA results was a peculiar pipetting mode of action of the Cobas e411, in which the analyzer uses a single pipette tip to first collect the anti-HAV reagent and then reuses the same pipette tip to collect the patient’s serum/plasma sample, which consequently causes the permanent contamination of the original sample with HAV RNA. In contrast, both the Vitros 3600 and the ARCHITECT use separate pipette tips to collect anti-HAV reagents and the sample. Such a unique and potentially harmful Cobas e411 pipetting approach is not disclosed in the instrument’s manual, and, consequently, for years, the laboratory was unaware of this important instrument drawback. The main reason that occasional HAV RNA false-positive results were not encountered earlier is that between 2012 and 2020, the Cobas e411 was used only as a backup analyzer, mainly for the resolution of initially indeterminate anti-HAV Vitros 3600 results, and, consequently, only 1 to 3 patients per year had HAV RNA-positive results that were inconsistent with patients’ anti-HAV serological profiles and/or medical histories. In late 2020, when the Cobas e411 become the frontline analyzer for anti-HAV serology, the number of suspicious HAV RNA false-positive results increased substantially to 10 per month, prompting immediate close inspection and a detailed, step-by-step review of all of the testing protocols that were being used in our laboratory. This finally resulted in the identification of the source and mode of the HAV RNA contamination that was causing the occasional HAV RNA false-positive results. Finally, we stopped using the Cobas e411 for anti-HAV serology in October of 2020.In conclusion, we describe occasional HAV RNA false-positive results that were detected during reflex molecular testing and linked with the preceding testing of serum samples for anti-HAV antibodies using an automated immunoassay analyzer. The peculiar pipetting procedure of this analyzer with a single pipette tip to collect both anti-HAV reagents and the sample contaminated the original patient sample with the HAV RNA that was present in the immunoassay’s reagents, thereby resulting in HAV RNA false positivity during reflex molecular testing. Manufacturers of automated immunoassay analyzers and corresponding assays should strongly consider the occasional need for reflex testing from preprocessed samples and design their products to prevent the contamination of processed samples, either by using separate pipette tips when collecting reagents and samples or by using recombinant viral proteins instead of inactivated whole viruses  as antigens in their immunoassays . If a tehttps://www.rivm.nl/en/havnet); (iv) additional follow-up serum and/or stool samples were collected from all individuals with suspected false-positive HAV RNA results; (v) frequent PCR-contamination checks of various laboratory surfaces were performed; (vi) several previously HAV RNA-negative samples and blank water samples were included in each PCR run; (vii) several serum samples were obtained from healthy anti-HAV and HAV RNA-negative controls and tested for HAV RNA; and (viii) multiple rounds of HAV RNA external quality assessment (EQA) control panels  were purchased and tested for HAV RNA.The following measures were taken to identify the potential source of the HAV RNA contamination that was leading to sporadic HAV RNA false-positive results: (i) all questionable samples were tested for HAV RNA in parallel using several commercial PCR assays, namely, the artus HAV RT-PCR Kit , then LightCycler HAV Quantification Kit (Roche Diagnostics), and the AltoStar HAV RT-PCR Kit , strictly following the manufacturers’ instructions, as well as the “in-house” HAV RNA PCR test ; (ii) HAA phylogenetic tree was inferred from all of the laboratory-generated HAV sequences from 2012 to 2020 using PhyML 3.0, with model selection occurring in accordance with the Bayesian information criterion .