338 results about "Reverse transcriptase" patented technology

The invention provides a method for preparing a chicken immortalized cell line. The method comprises the following steps: constructing a plasmid system of conditional over-expression TERT (Telomerase Reverse Transcriptase), carrying out ovarian injection and breeding to obtain a transgenic chicken of the conditional over-expression TERT, and carrying out separation and induction to obtain the chicken immortalized cell line. The obtained chicken immortalized cell line has the characteristics of primary cells and is susceptible to viruses; the method can be used for virus reproduction and detection, influenza virus vaccine production and the like, can obtain immortalized cell lines of all organs and all parts of the chicken in a short time, and has a good application prospect in the vaccine industry.

This invention relates to recombinant nucleic constructs comprising a DNA binding domain, an endonuclease and a reverse transcriptase and methods of use thereof for modifying nucleic acids in plants.

Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and / or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.

The invention belongs to the field of biological detection, and discloses an exosome RNA detection composition based on positive and negative targets and a use method thereof, the composition comprises two positive targets and one negative target: the positive target A is miRNA stably expressed in exosome and is used for reflecting the expression level of small RNA; the positive target B is typical mRNA and is used for evaluating the quality of the long-fragment RNA; the negative target C is bacterial 16S rRNA and is used for identifying bacterial nucleic acid pollution in a sample. The method combines III-generation reverse transcriptase and HotStart Taq enzyme to construct a qPCR system, has high sensitivity and specificity, and is suitable for rapid detection of trace RNA. Whether the exosome RNA is suitable for downstream analysis or not can be judged by acquiring the Cq values of the three types of targets and setting a reference range. According to the invention, the integration of quality control, quantification and pollution identification of the exosome RNA is realized.

The present disclosure provides systems, compositions, methods, and kits, for guided DNA synthesis and genome engineering. Particularly, the present disclosure provides systems, compositions, methods, and kits comprising a defense-associated reverse transcriptase (DRT), or a nucleic acid encoding thereof, and an engineered target nucleic acid comprising one or more sequence of interest, or a nucleic acid encoding thereof.

The invention belongs to the technical field of molecular diagnosis, and particularly discloses a high-risk HPV16 rapid detection system based on RPA-CRISPR / Cas12a, which is prepared by the following steps: (1) mixing a nucleic acid lysis solution containing Tris (pH = 8.0), NaHCO3, polyvinylpyrrolidone and Tween-20 with Proteinase K to treat a sample, and performing pyrolysis at room temperature for 5 minutes to obtain a nucleic acid template; (2) an RPA reaction system contains an RNA inhibitor, a reverse transcriptase and an RPA reaction essential enzyme, and after the template and the starting agent in the step (1) are added, RPA amplification is carried out at 37 DEG C; (3) dropping the reaction liquid on a colloidal gold test strip subjected to position exchange and transformation of a sample pad and a water absorption pad, and carrying out result interpretation; and (4) adding the RPA product into a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reaction system containing the LbCas12a protein, the specific gRNA and the probe. According to the scheme, a rapid, high-sensitivity and cross-reaction-free detection system of the HPV16 is realized by optimizing a nucleic acid lysis solution formula, designing a specific primer and gRNA and modifying a test strip for detection.

Telomerase reverse transcriptase (TERT) expression enhancing compounds, and methods for using the same, are provided. These compounds and methods find use in a variety of applications in which increased expression of telomerase reverse transcriptase is desired.

The present invention provides MMLV reverse transcriptase enzymes with increased thermal stability as compared with wild type MMLV and AMV reverse transcriptases. The improved thermal stability allows for reverse transcription of RNA to cDNA at temperatures above 37° C., thereby reducing error rates introduced during cDNA synthesis. As a result, the reverse transcriptases of the invention allow for increased accuracy in the determination of transcriptomes of living organisms.

The invention discloses a polypeptide ligand for improving the thermal stability of MMLV reverse transcriptase and application of the polypeptide ligand, and relates to the technical field of biology. The invention specifically discloses a polypeptide ligand with an amino acid sequence as shown in SEQ ID NO: 1. The polypeptide ligand can obviously improve the thermal stability of MMLV RT. After being combined with the polypeptide ligand, the MMLV RT can effectively maintain the activity of the MMLV RT at 45-60 DEG C and maintain good activity in a freeze-thaw cycle, and the Ct value change is small after the MMLV RT is placed at 37 DEG C for more than 15 days, so that the polypeptide has a very good application prospect in industrial production and commercial application.

Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and / or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.

The invention provides methods and compositions for treating medical disorders, such as cancer, an autoimmune disorders, and / or neurological disorders, and modulating LINE1 reverse transcriptase and / or HERV-K reverse transcriptase using islatravir or a related compound.

Described herein are fusion proteins comprising intein polypeptides and methods of using these proteins. The fusion proteins described herein may include a Cas12a polypeptide and an intein polypeptide or a polypeptide of interest (e.g., a reverse transcriptase polypeptide) and an intein polypeptide. Also described herein are compositions and systems for modifying or editing a target nucleic acid.

The present invention relates to a prime editing system with enhanced correction precision and a use thereof. More specifically, the present invention relates to a recombinant vector for prime editing with improved precision, comprising: a prime editor expression cassette including a mutant Cas9 (R221K, N394K, D840A), a nucleocapsid protein, and an RNaseH domain-deleted mutant MMLV reverse transcriptase (D200N, T306K, W313F, T330P, L603W); and a pegRNA (prime editing guide RNA) expression cassette transcriptionally regulated by a U6 composite promoter.

A gene editing system comprising (a) a fusion polypeptide comprising an RNA-guided nuclease and a reverse transcriptase, or a nucleic acid encoding the fusion polypeptide, and (b) an RNA molecule comprising a guide RNA and a reverse transcription donor RNA, or a nucleic acid encoding the RNA molecule. Also provided herein are methods of using the gene editing system for modifying target genes of interest.

The present disclosure provides evolved and engineered reverse transcriptase variants and Cas9 variants with improved properties (e.g., improved editing efficiency when used in a guided editor context). The disclosure also provides fusion proteins comprising the reverse transcriptase variants and Cas9 variants described herein, including, for example, a guided editor. The disclosure also provides polynucleotides encoding the reverse transcriptase variants, Cas9 variants, and guide editors provided herein, as well as vectors comprising such polynucleotides. The disclosure also provides pharmaceutical compositions and cells comprising the reverse transcriptase variants, Cas9 variants, and a guided editor described herein. The present disclosure also provides methods and uses relating to the reverse transcriptase variants, Cas9 variants, and guide editors described herein.

Provided is a reverse prime editing system, which is used for achieving site-specific and precise small-fragment DNA editing upstream of a target site. In the reverse prime editing system, a targeted strand nickase (such as D10A-Cas9) is used to cleave a nick on a targeted strand, thereby enabling precise small-fragment DNA base insertion, deletion and substitution upstream of the target site under the action of a reverse transcriptase.

The invention discloses application of novel reverse transcriptase and related fusion protein of the novel reverse transcriptase in plant-guided editing. Candidate RT protein is obtained through systematic mining from a public database, novel reverse transcriptase cl1 with activity is screened out in combination with a fluorescence report system, and a guided editing system suitable for plants is constructed. The system tests in wheat protoplast, and verifies the editing activity of cl1 to a plurality of endogenous targets. Further combining with protein structure prediction and rational design point mutation optimization, the cl1 mutant with higher editing efficiency is obtained, and the method can be applied to accurate and effective editing of plant genomes and plant breeding and improvement.

The invention discloses a reverse transcriptase, wherein a coding sequence of the reverse transcriptase is derived from a Rattus norvegicus genome. The protein sequence of the protein comprises a protein sequence of a wild type Rattus norveicus, or a protein sequence which is subjected to engineering modification on the basis of the wild type Rattus norveicus, or a protein sequence which is subjected to engineering modification on the basis of the wild type Rattus norveicus. Furthermore, the protein sequence of the gene is as shown in any one of SEQ No.1-55. In a rice genome site and a human HEK 293T cell genome site, the system shows efficient editing efficiency of a guide editor. The invention provides a series of efficient reverse transcriptase components which can be carried on a guide editing system, important bottom-layer technical support is provided for research of point mutation in a genome, creation of a special site disease model and correction of genetic mutation sites, and the reverse transcriptase component has a wide application prospect in the field of precision medicine and precision agriculture breeding.

The present disclosure provides compositions and methods for inserting heterologous payload sequences into a target-site in a host cell genome. The compositions and methods use non-LTR retrotransposon reverse transcriptase proteins that bind template RNAs comprising a payload sequence that encodes a protein or regulatory RNA. The template RNA can comprise modified uridines that are not cleavable by a ribozyme. The incorporation of modified uridines increases the efficiency of integration and expression of the payload sequence and decreases cellular toxicity.

The present disclosure relates to crystalline and solvate forms of nucleoside reverse transcriptase translocation inhibitor (NRTTI) and pharmaceutical compositions thereof for use in the treatment and prevention of retroviridae viral infections, including infections caused by HIV virus.

The invention belongs to the technical field of biology, and relates to a pathogen detection technology based on a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, in particular to a tubercle bacillus detection and activity determination method based on fluorescent T7-CRISPR. The first purpose is to provide a reagent combination for detecting viable tubercle bacillus, and the reagent combination comprises a) a T7 reverse transcription reaction reagent and b) a CRISPR-Cas13a reaction system; a) the T7 reverse transcription reaction reagent comprises a T7 reverse transcription primer and a T7 reverse transcriptase; and b) the CRISPR-Cas13a reaction system comprises specific crRNA, a fluorescence labeled ssRNA reporter, a reaction buffer solution, a Cas13a protein, RNase-Free H2O and a T7 reverse transcriptase. The invention also provides application of the reagent combination in detection of viable tubercle bacillus. The primer combination disclosed by the invention has the advantages of strong specificity and high sensitivity in detection application, can realize rapid detection and visual detection, and is low in detection cost, simple and convenient to operate and suitable for large-scale clinical application.

The disclosure provides Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) mutants. The disclosure as provides suitable amino acid positions in MMLV RTase for mutagenesis and methods and kits for using MMLV RTase mutants to synthesize cDNA from RNA templates.

This invention relates to recombinant nucleic constructs comprising a DNA binding domain, an endonuclease and a reverse transcriptase and methods of use thereof for modifying nucleic acids in plants.

The invention provides a retrotransposase, which comprises a target DNA binding structural domain containing a zinc finger binding motif, a reverse transcriptase structural domain and an endonuclease structural domain, and can be used for reversely transcribing RNA (Ribonucleic Acid) into DNA. The amino acid sequence of the retrotransposase is shown as any one of SEQ ID No.1 to 6 or SEQ ID No.32 to 43 or SEQ ID No.68 to 71 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% identity with the amino acid sequence of any one of SEQ ID No.1 to 6 or SEQ ID No.32 to 43 or SEQ ID No.68 to 71. The invention also relates to a system for modifying DNA. The system comprises the retrotransposase provided by the invention or a nucleic acid encoding the retrotransposase provided by the invention; and a donor RNA or a nucleic acid encoding the donor RNA, the donor RNA comprising: a sequence that binds to the retrotransposase and a heterologous sequence.

The present disclosure provides a recombinant reverse transcriptase with improved processivity and the use thereof. In one embodiment, the recombinant reverse transcriptase is a fusion protein comprising a reverse transcriptase and a cold shock protein.

The invention discloses an MMLV reverse transcriptase mutant. The invention provides an MMLV reverse transcriptase mutant, and compared with a wild type MMLV reverse transcriptase amino acid sequence, the mutant mutates at a plurality of sites such as a 62 site and the like, and the mutant has the characteristics that the template conversion performance is improved, the non-template basic group addition performance is improved or the heat resistance is improved. The MMLV reverse transcriptase mutant with improved conversion performance compared with a wild type template is screened out, and the MMLV reverse transcriptase mutant also has improved non-template base addition performance or thermal stability; the screened mutant is suitable for RNA (Ribonucleic Acid) sequencing, such as Smart-Seq, nanopore sequencing, 5 'RACE (Rapid Amplification of cDNA Ends) and the like, adopting a library preparation method based on template conversion, and can also be applied to RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) and the like.

Disclosed herein is a chimeric nucleic acid including a human telomerase reverse transcriptase (hTERT) enhancer or a fragment thereof and an SV40 enhancer or a fragment thereof and methods of using the same.

The present disclosure provides Moloney murine leukemia virus (MMLV) reverse transcriptase (RTase) variants. The present disclosure further provides amino acid positions for mutagenesis of MMLV RTase as well as nucleic acids, kits, compositions, fusion proteins, and methods including MMLV RTase variants.

The present invention provides a method for screening for a telomerase reverse transcriptase (TERT) phosphorylation inhibitor using TERT or a fragment thereof, and a TERT kinase.

The application discloses a method for synthesizing reverse transcriptase: obtaining a target reverse transcriptase expression sequence based on database primary screening; obtaining a candidate reverse transcriptase expression sequence by using a fluorescent reporter system to rescreen the target reverse transcriptase expression sequence; and determining a synthetic reverse transcriptase expression sequence by using the fluorescent reporter system after optimizing the candidate reverse transcriptase expression sequence. The application further screens a plurality of natural or engineered small reverse transcriptases in view of the bottleneck of the prior art, and verifies the applicability of the reverse transcriptases in prime editing. The technology is expected to break through the application limitation of the existing prime editing system, and provide more efficient and safer precise editing tools for the fields of genetic disease treatment, agricultural breeding and synthetic biology.