48 results about "RNA polymerase III" patented technology

The invention discloses a method for improving the efficiency of CRISPR mediated homologous recombination. The method includes the steps of: inhibiting the expression of Lig4, DNA-PK and XRCC6 by shRNA so as to inhibit the non-homologous end joining (NHEJ) repair pathway; conducting fusion expression on sgRNA and shRNA at targeting genome specific positions to form shRNA-sgRNA polycistron; and placing the polycistron at the RNA polymerase II or RNA polymerase III promoter downstream. The carrier constructed by the method provided by the invention has a concise structure and is convenient to use, also enhances the homologous recombination efficiency by dozens of times, greatly expands the function and application of the CRISPR carrier, and is a good tool for functional gene and signal pathway research, medical research, and recombinant protein expression.

The invention relates to an infectious arenavirus particle that is engineered to contain a genome with the ability to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells. One or more of the four arenavirus open reading frames glycoprotein (GP), nucleoprotein (NP), matrix protein Z and RNA-dependent RNA polymerase L are removed or mutated to prevent replication in normal cells but still allowing gene expression in arenavirus vector-infected cells, and foreign genes coding for an antigen or other protein of interest or nucleic acids modulating host gene expression are expressed under control of the arenavirus promoters, internal ribosome entry sites or under control of regulatory elements that can be read by the viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II or RNA polymerase III. The modified arenaviruses are useful as vaccines and therapeutic agents for a variety of diseases.

The invention belongs to the technical field of gene engineering, particularly relates to a rubber tree RNA (ribonucleic acid) polymerase type III promoter, in particular to a rubber tree U6 gene promoter proHbU6.6 and further discloses a cloning method and an application of the rubber tree U6 gene promoter proHbU6.6. The rubber tree RNA polymerase type III promoter, namely the rubber tree endogenous U6 promoter proHbU6.6 is obtained by cloning in a Para rubber tree for the first time; the promoter is a rubber tree endogenous RNA polymerase type III promoter, has high transcriptional activity,and can drive expression of downstream sgRNA (small guide ribonucleic acid); the activity of the promoter and feasibility of the promoter applied to a rubber tree CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated nuclease 9) gene editing system are demonstrated by instantaneous conversion of rubber tree protoplast; and CRISPR / Cas9 mediated rubber tree genometarget editing is achieved.

The invention belongs to the technical field of gene engineering, and relates to a rubber tree RNA polymerase III type promoter, in particular to a rubber tree U6 gene promoter proHbU6.8. The invention further discloses a cloning method and application of the promoter. The rubber tree RNA polymerase III type promoter-rubber tree endogenous U6 promoter proHbU6.8 is obtained through cloning in a Brazil rubber tree for the first time. The promoter is the rubber tree endogenous RNA polymerase III-type promoter, the promoter has high-efficiency transcriptional activity, the expression of downstreamsgRNA can be driven, the activity of the promoter and the application feasibility of the promoter in a rubber tree CRISPR / Cas9 gene editing system are verified through transient transformation rubbertree protoplasm, and CRISPR / Cas9-mediated rubber tree genome targeted editing is achieved.

In a first aspect, the invention provides expression vectors comprising: (a) a first RNA polymerase III promoter operably associated with a first RNA polymerase III termination signal and (b) a second RNA polymerase III promoter operably associated with a second RNA polymerase III termination signal, wherein the first and second RNA polymerase III promoters are oriented to promote bidirectional transcription of an insert disposed between the first and the second RNA polymerase III termination signals. In other aspects, the invention provide methods for using these expression vectors for inhibiting expression of target genes, for determining the effect of siRNAs on biological processes, and for identifying siRNAs that affect biological processes.

The present disclosure provides expression vectors containing a nucleic acid encoding an RNA polymerase III promoter, a ribozyme, a CRISPR-Cas9 single guide RNA, and an RNA polymerase III terminator, where the ribozyme is 5′ to the CRISPR-Cas9 single guide RNA, as well as ribonucleic acids encoded thereby. Further provided are fungal cells containing an expression vector described herein, as well as methods of fungal genome engineering through use of an expression vector described herein.

The invention relates to a nucleic acid comprising a nucleotide sequence encoding a tRNA orthogonal to a eukaryotic cell, said nucleotide sequence operably linked to a promoter capable of directing transcription by eukaryotic RNA polymerase III. The invention also relates to methods for incorporating unnatural amino acids in eukaryotic cells using same.

The invention belongs to the technical field of gene engineering, particularly relates to an asparagus RNA polymerase III type promoter, more particularly relates to an asparagus U6 gene promoter AspU6P3, and further discloses a cloning method and application of the asparagus U6 gene promoter AspU6P3. The asparagus RNA polymerase III-type promoter, namely the asparagus endogenous U6 promoter AspU6P3, is cloned from asparagus (aka the king of vegetables) cultivated species for the first time; the promoter is the asparagus endogenous RNA polymerase III-type promoter and has efficient transcriptional activity, and can drive expression of downstream sgRNA; in addition, The activity of the endogenous U6 promoter AspU6P3 and the feasibility of the endogenous U6 promoter AspU6P3 applied to an asparagus CRISPR / Cas9 gene editing system are verified through an asparagus stable genetic transformation system, and CRISPR / Cas9 mediated asparagus genome targeted editing is realized for the first time.

Expression constructs comprising at least two different RNA polymerase III promoters, wherein each promoter is operably linked to a nucleic acid sequence encoding an RNA effector molecule, are disclosed herein. Further provided are expression constructs comprising multiple polymerase III promoters operably linked to sequences encoding short hairpin RNA molecules, which may comprise single and / or multiple fingers. The provided constructs are useful for in vivo delivery of RNA molecules effective in gene silencing, including of viral genes including HBV and HCV.

The present invention relates to an isolated DNA molecule from a thermophilic bacterium which encodes a DNA polymerase III-type enzyme subunit. Also encompassed by the present invention are host cells and expression system including the heterologous DNA molecule of the present invention, as well as isolated replication enzyme subunits encoded by such DNA molecules. Also disclosed is a method of producing a recombinant thermostable DNA polymerase III-type enzyme, or subunit thereof, from a thermophilic bacterium, which is carried out by transforming a host cell with at least one heterologous DNA molecule of the present invention under conditions suitable for expression of the DNA polymerase III-type enzyme, or subunit thereof, and then isolating the DNA polymerase III-type enzyme, or subunit thereof.

The invention belongs to the technical field of gene engineering, particularly relates to a rubber tree RNA polymerase III-type promoter, more specifically relates to a rubber tree U6 gene promoter proHbU6.1, and further discloses a cloning method and applications thereof. The invention clones and obtains the rubber tree RNA polymerase III-type promoter-rubber tree endogenous U6 promoter proHbU6.1in hevea brasiliensis for the first time, the promoter is rubber tree endogenous RNA polymerase III-type promoter, the promoter has high-efficiency transcription activity and can drive downstream sgRNA expression, the activity and the feasibility of the promoter for application in rubber tree CRISPR / Cas9 gene editing system are verified by transient transformation of rubber tree protoplasts, andCRISPR / Cas9 mediated rubber tree genome targeted editing is realized.

Expression constructs comprising at least two different RNA polymerase III promoters, wherein each promoter is operably linked to a nucleic acid sequence encoding an RNA effector molecule, are disclosed herein. Further provided are expression constructs comprising multiple polymerase III promoters operably linked to sequences encoding short hairpin RNA molecules, which may comprise single and / or multiple fingers. The provided constructs are useful for in vivo delivery of RNA molecules effective in gene silencing, including of viral genes including HBV and HCV.

The invention belongs to the technical field of gene engineering, particularly relates to a potato U6 RNA polymerase III type promoter, more particularly relates to StU64-1 and StU68-1 gene promoters, and further discloses a cloning method and application thereof. Two potato RNA polymerase III type promoters StU64-1 and StU68-1 are obtained by cloning in potatoes, have efficient transcriptional activity and can drive downstream sgRNA expression, and the activities of the two promoters and the feasibility of applying the promoters to potato CRISPR / Cas9 gene editing are respectively verified by virtue of an instantaneous transformation system of nicotiana benthamiana leaves and a stable transformation system of potatoes, and the CRISPR / Cas9 guided potato genome editing is realized. In the technical field of transgenosis, the promoters are not only suitable for potatoes, but also can be applied to other solanaceae crops such as tobacco.

The present invention relates to an isolated DNA molecule from a thermophilic bacterium which encodes a DNA polymerase III-type enzyme subunit. Also encompassed by the present invention are host cells and expression system including the heterologous DNA molecule of the present invention, as well as isolated replication enzyme subunits encoded by such DNA molecules. Also disclosed is a method of producing a recombinant thermostable DNA polymerase III-type enzyme, or subunit thereof, from a thermophilic bacterium, which is carried out by transforming a host cell with at least one heterologous DNA molecule of the present invention under conditions suitable for expression of the DNA polymerase III-type enzyme, or subunit thereof, and then isolating the DNA polymerase III-type enzyme, or subunit thereof.

The invention discloses a bicistronic co-expression gene transfer body and a preparation method. The gene sequence of the gene transfer body is SEQ ID NO: 17. The gene transfer body comprises bovine matrix attachment regions (MARs), an RNA polymerase promoter III (U6), short hairpin RNA (shRAN) targeting the third exon of bovine myostatin (MSTN), a CMV promoter, a bovine follistatin (FSTN) and an SV40polyA signal region from the five prime end to the three prime end in order. The gene transfer body provided by the invention doesn't contain a vector backbone sequence and is a clean and safe gene transfer; and the gene transfer body combines the MAR with a knock-down vector for the first time and can effectively enhance the knock-down effect of the vector. Besides, the gene transfer body achieves the purpose of conducting knock-down and over-expression simultaneously by independently starting double promoters, and provides new ideas and paths for improving the characteristics (such as economic characteristics) of double-gene / multi-gene control.

Fusion promoters are described that combine a RNA polymerase III basal promoter and regulatory elements from RNA polymerase II regulatory regions, and which provide specific regulation of expression from the promoter. Such fusion promoters are useful, for example, for expressing RNAi agents in vivo.

The invention provides an application of RNA polymerase III transcribed AtR8 long noncoding RNA (lncRNA) to arabidopsis thaliana, relates to an application of long noncoding RNA to the arabidopsis thaliana, and aims to solve the problem of an arabidopsis thaliana seed germinating physiology mechanism not clear in a molecular layer, and particularly solve the problem of the seed germinating mechanism with AtR8 long noncoding RNA. The situation that the AtR8 lncRNA is RNA polymerase III transcribed long noncoding RNA in response to ABA stress in an arabidopsis thaliana seed germinating course isfound. The situation that the AtR8 lncRNA participates in the arabidopsis thaliana seed germinating course under ABA stress is proved. Under the normal growth condition, the AtR8 lncRNA participatesin growth and development of siliques, rosette leaves and seeds of arabidopsis thaliana. Through analysis, the AtR8 lncRNA possibly plays an important role in normal development of the arabidopsis thaliana and seed germination under ABA stress through influencing the expression of ABA signal pathway key genes ABI5 and EM6. The RNA polymerase III transcribed AtR8 long noncoding RNA (lncRNA) in arabidopsis thaliana disclosed by the invention is applied to the field of the arabidopsis thaliana.

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats) / Cpf1 gene editing system as well as a construction method and the application thereof in gibberella. The system is a skeleton plasmid pCpf1Ff-DR, and comprises an Fncpf1 gene, a functional crRNA gene, a promoter pGPD, a promoter pTRPC, a strong RNA polymerase III type promoter 5srRNA and a hygromycin selection marker hph. The system is high in efficiency, strong in universality, easy to operate and capable of being rapidly applied to filamentous fungus gibberella, and the gene editing system can be used for achieving knockout of the bicarpin gene cluster as long as 17kb in the gibberella. According to the method, large-fragment gene knockout is achieved in gibberellin for the first time, it is found for the first time that knockout of the bicarpin gene cluster is beneficial to increase of the yield of gibberellin, and the method is expected to be further used for transforming gibberellin industrial bacteria, shortening the time of bacterial strain upgrading and providing powerful guarantee for increasing the yield of gibberellin and increasing economic benefits.

The invention discloses a broad-spectrum anti-apoptosis rhabdovirus expression vector, and the broad-spectrum anti-apoptosis effect is achieved through the expression of the caspase-1 consensus sequence of target Spodoptera frugiperda and Trichoplusia ni insect cells by the vector. The rhabdovirus vector comprises a section of specific DNA sequence, the DNA sequence comprises the siRNA sequence ofthe consensus sequence of the target Sf-caspase-1 and Tn-caspase-1 transcribed by an RNA polymerase III promoter; the recombinant virus of the vector expresses double-strand small RNA in a host cell,the caspase-1 encoded by the silent host cell is interfered through the RNA, so that the apoptosis of the host cell is inhibited, and the expression level of a foreign protein is obviously improved.The broad-spectrum anti-apoptosis rhabdovirus expression vector can be used in the industrial production of protein preparations and vaccines.

RNA interference (RNAi) has become a powerful tool for blocking gene expression in mammals and mammalian cells. This approach requires the delivery of small interfering RNA (siRNA) either as RNA itself or as DNA, using an expression plasmid or virus and the coding sequence for small hairpin RNAs that are processed to siRNAs. Current expression systems for the production of siRNA in vivo rely on RNA polymerase III promoters. These are difficult to regulate and leave most of the RNA in the nucleus of the cell where it is inactive for RNA interference. The invention provides a DNA cassette for the cloning of small hairpin sequences which permit their expression and processing using RNA polymerase II. This system enables efficient transport of the pre-siRNAs to the cytoplasm where they are active and permits the use of regulated and tissue specific promoters for gene expression.

Methods and means are provided to increase the efficiency of gene silencing when using dsRNA sequences which have a stem length shorter than about 200 base pairs by providing chimeric genes encoding such dsRNA sequences with a promoter recognized by DNA dependent RNA polymerase III comprising all cis-acting promoter elements which interact with DNA dependent RNA polymerase III.

The invention relates to a fraxinus mandshurica U6 gene promoter proFmU6.7 as well as cloning and application thereof. According to the invention, the fraxinus mandshurica RNA polymerase III type promoter, namely the fraxinus mandshurica endogenous U6 gene promoter proFmU6.7, is successfully cloned for the first time, an activity detection carrier of the fraxinus mandshurica U6 gene promoter is successfully constructed, and through instantaneous transformation of fraxinus mandshurica seedlings and GUS dyeing verification, the promoter is proved to have efficient transcriptional activity. Experiments prove that the fraxinus mandshurica U6 gene truncated promoter proFmU6.7.4 has starting activity and is successfully applied to a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9) system. According to cloning of the fraxinus mandshurica U6 gene promoter proFmU6.7 and expression analysis of promoter activity, an efficient promoter sequence is provided for research on transformation of fraxinus mandshurica and related plants, and efficient and accurate germplasm innovation and variety genetic improvement of fraxinus mandshurica can be achieved.

In the present invention, by amplifying a segment of Rhodosporidium toruloides DNA sequence and performing biological function verification, the obtained genus Rhodosoporidium, Sporidiobolus, and Saccharomyces ( Sporobolomyces) and Rhodotorula (Rhodotorula), RNA polymerase type III promoters for non-coding RNA transcription, genetic engineering operations and strain improvement, the nucleotide sequence of which is SEQ ID NO:1. The present invention also relates to a method for constructing genetically engineered strains of the genus Rhodosporidium, Saccharomyces genus and Rhodotorula genus and the corresponding strains containing the DNA sequence expression cassette or recombinant vector, and using related elements.

The invention relates to a method for designing a siRNA (Small Interfering Ribonucleic Acid) molecule resisting RNA virus. The method comprises the following steps of: (1) selecting RNA polymerase, which is in RNA virus, has a conserved sequence and is RNA-dependent, as a target of siRNA; (2) utilizing siRNA design software to generate small RNA molecules resisting the RNA virus; (3) screening theoretically efficient siRNA for experiment research according to a siRNA design rule; and (4) adopting expression plasmid containing RNA polymerase III promoter U6 as an antiviral application form of the small RNA molecules. The method is applied to select an antiviral target sequence, so that the probability of obtaining efficient and antiviral small RNA molecules can be improved obviously, the selection range of an RNA interference target point is greatly reduced, the working efficiency is improved, and the cost for RNA interference technology research and application is obviously reduced.