Disclosed herein are engineered guide RNAs, constructs for forming engineered guide RNAs, pharmaceutical compositions thereof, methods of making the engineered guide RNAs, and methods of treating or preventing a diseases and disorders of a subject by administering one or more of the engineered guide RNAs or the constructs for forming the engineered guide RNAs.

A method of cyclizing an L-RNA aptamer by modifying the aptamer with a 3′ azide and a 5′ alkyne group and using click chemistry reaction-based method. The cyclized L-RNA aptamers have improved binding properties and favour more in vitro/cell applications. Also disclosed is an L-oligonucleotide aptamer having linked ends.

Programmable guide RNAs (gRNAs) play a central role in the CRISPR revolution sweeping biology and medicine by directing the function of a Cas protein effector to a target gene of choice. To achieve programmable control over regulatory scope, the activity of a conditional guide RNA (cgRNA) depends on the presence or absence of an RNA trigger, allowing for cell-selective regulation of CRISPR/Cas function. Unlike a standard gRNA, a cgRNA is programmable at multiple levels, with the target-binding sequence controlling the target of Cas activity (edit, silence, induce, or bind a gene of choice) and the trigger binding sequence controlling the scope of Cas activity. cgRNA mechanisms that are allosteric allow for independent design of the target and trigger sequences, providing the flexibility to select the regulatory target and scope independently. Disclosed herein are allosteric cgRNA mechanisms for both ON.fwdarw.OFF logic (conditional inactivation by an RNA trigger) and OFF.fwdarw.ON logic (conditional activation by an RNA trigger). Allosteric cgRNAs enable restriction of CRISPR/Cas function to a desired cell type, tissue, organ, or disease state. Allosteric cgRNAs provide a versatile platform for cell-selective and tissue-selective research tools, biotechnologies, diagnostics, and therapeutics.

The invention relates to a delivery vector for the delivery of a single-stranded nucleic acid. Said vector is a closed circular polynucleotide comprised of at least three sections, two of which have sufficient complementarity to form a duplex, and an intervening sequence containing the single-stranded nucleic acid to be delivered. Said duplex includes a recognition sequence for a targeted nuclease such that under appropriate conditions the single-stranded nucleic acid is released.

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5′ to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.

The present disclosure relates to a recombinant nucleic acid molecule of the transcriptional circular RNA and its application in protein expression. Specifically, the present disclosure relates to a recombinant nucleic acid molecule of the transcriptional circular RNA, recombinant expression vector, pre-circularized RNA, circular RNA, recombinant host cell, pharmaceutical composition and protein preparing method. The transcription product of the recombinant nucleic acid molecule in this present disclosure is a circular RNA which containing specific IRES element. IRES element can increase the protein expression level of circular RNA in eukaryotic cells, achieve efficient and persistent expression of protein. It has important application value in many fields like: Preparation of mRNA infectious disease vaccines, therapeutic mRNA tumor vaccines, mRNA-based dendritic cell tumor vaccines, mRNA-based gene therapy, mRNA-based chimeric antigen receptor T cell therapy, and protein supplement therapy.

The present invention provides an enrichment method for a gene target region, the method comprising: (1) amplifying, by means of a specific probe, fragmented DNA including a target region, and providing a captured extension product, wherein the specific probe comprises a sequence that is complementary to the target region of the fragmented DNA, and a 3′-end nucleotide and a 5′-end nucleotide of the probe are both modified; and (2) adding a ligase to the captured extension product provided in step (1), and providing a ligation product. The invention further provides an enrichment system for a gene target, the system being applicable to the enrichment method for a gene target region provided in the present invention.

Disclosed herein are circular RNA s and transfer vehicles, along with related compositions and methods of treatment. The circular RNAs can comprise group I intron fragments, spacers, an IRES, duplex forming regions, and/or an expression sequence, thereby having the features of improved expression, functional stability, low immunogenicity, ease of manufacturing, and/or extended half-life compared to linear RNA. Pharmaceutical compositions comprising such circular RNAs and transfer vehicles are particularly suitable for efficient protein expression in immune cells in vivo. Also disclosed are precursor RNAs and materials useful in producing the precursor or circular RNAs, which have improved circularization efficiency and/or are compatible with effective circular RNA purification methods.

The present disclosure belongs to the technical field of bio-medicine, particularly, the present disclosure relates to a tissue-specifically expressed circular RNA molecule, a cyclization precursor RNA molecule, a recombinant nucleic acid molecule, a recombinant expression vector, a recombinant host cell, a composition, a pharmaceutical preparation and application thereof in preparation of a drug for preventing or treating diseases, as well as a method for preventing or treating diseases. The circular RNA molecule provided by the present disclosure operably links a coding region to an expression regulatory element, realizing specific high expression of a target polypeptide in target cells or target tissues, and low expression of the target polypeptide in non-target cells or non-target tissues, and having high tissue expression specificity, thereby providing a safe and effective treatment strategy for clinical targeted therapy of diseases such as tumors.

Disclosed herein are circular RNAs and transfer vehicles, along with related compositions and methods of treatment. The circular RNAs can comprise group I intron fragments, spacers, an IRES, duplex forming regions, and/or an expression sequence, thereby having the features of improved expression, functional stability, low immunogenicity, ease of manufacturing, and/or extended half-life compared to linear RNA. Pharmaceutical compositions comprising such circular RNAs and transfer vehicles are particularly suitable for efficient protein expression in immune cells in vivo. Also disclosed are precursor RNAs and materials useful in producing the precursor or circular RNAs, which have improved circularization efficiency and/or are compatible with effective circular RNA purification methods.