1346 results about "Base J" patented technology

The specification provides programmable base editors that are capable of introducing a nucleotide change and / or which could alter or modify the nucleotide sequence at a target site in mitochondrial DNA (mtDNA) with high specificity and efficiency. Moreover, the disclosure provides fusion proteins and compositions comprising a programmable DNA binding protein (e.g., a mitoTALE, a mitoZFP, or a CRISPR / Casp) and double-stranded DNA deaminase that is capable of being delivered to the mitochondria and carrying out precise installation of nucleotide changes in the mtDNA. The fusion proteins and compositions are not limited for use with mtDNA, but also may be used for base editing of any double-stranded target DNA.

The present disclosure provides, among other things, single sample multi-attribute methods of simultaneously identifying and / or quantifying RNA capping and tailing modifications. The present disclosure provides, among other things, oligonucleotides comprising RNA and DNA bases that are complementary to the 5' and 3' ends of RNA. Provided herein is a method of manufacturing RNA for therapeutic use having a quantified percentage of capped and untailed mRNA, and a specified poly A tail length and polydispersity. Also provided herein is a quality control assay for a manufacturing lot.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids or the modification of nucleic acids or proteins, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of nucleic acid programmable DNA binding proteins e.g., GeoCas9 or variants thereof, and effector domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing or protein modification are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of a GeoCas9 and effector domains, are provided.

The present disclosure provides methods for reducing sequencing errors comprising one or any combination of: (i) removing deaminated bases in any nucleic acid molecule throughout a library preparation workflow which includes immobilised splints which bind to the library, the use of a compaction oligonucleotide, optionally with an intervening sequence, formation of closed circular nucleic acids, creating gaps using glycosylase and lyase activities at positions with deaminated bases. The library may be sequenced using pairwise sequencing, e.g. with dark sequencing and / or sequencing using a multivalent labelled probe for the formation of an avidity molecule and soluble primer and polymerase. Method for sequencing concatemers in which the concatermers are contacted with polymerases, soluble primers and a multivalent labelled molecule which forms a complex with the polymerase. Detecting polymerase position and nucleobase bound to the polymerase in the complex. These methods generate higher quality base calls during downstream sequencing workflows.

The invention belongs to the field of gene engineering, and relates to a DNA binding protein based on TraC effect protein, a fusion protein with a cytosine / adenine base editing function, an editing system and nucleic acid molecules or constructs for encoding the DNA binding protein and the fusion protein. The invention also relates to complexes and kits for gene editing comprising the fusion proteins of the invention or the nucleic acid molecules encoding them. The base editing tool disclosed by the invention can realize efficient, accurate and safe base editing in cells of various eukaryotes such as animals and plants. The unique characteristics of the base editing tool enrich the types of existing base editing tools, and the base editing tool has wide application prospects.

The invention provides a fusion protein with sequence preference and a gene editing system, and relates to the technical field of gene editing. The invention discloses that the key 48th amino acid for binding TadA-8e and substrate DNA influences the overall editing efficiency, editing accuracy and sequence preference of ABE8e for the first time; through rational design, a TadA-8e part in ABE8e is modified, saturation mutation is designed at a key 48th amino acid for combining the TadA-8e and a substrate DNA, and YAY high-precision ABE with sequence preference is developed. According to the method, A in a sequence YAY (Y = T or C) in an editing window can be accurately edited into G, other A is not edited or only low editing is generated, the problem of'adjacent base 'mutation is avoided, and potential safety hazards caused by wrong mutation are reduced.

Nucleic acid molecules, compositions, recombinant AAV (rAAV) particles, kits, and methods are described herein for delivering a base editor (or “nucleobase editor”) to cells, e.g., via AAV vectors. In particular, the disclosure provides compositions, methods, and uses for delivery of adenine base editors and cytosine base editors in a single AAV vector (or genome). Further described herein are improved AAV vectors containing size-minimized regulatory components that enable, e.g., the packaging of base editors. Provided herein are methods and compositions for delivering base editor proteins to a cell or tissue in a single recombinant AAV (rAAV) vector. Contemplated herein are improved methods and compositions for delivering these base editors in vivo, in a single rAAV particle. Further provided herein are base editors and compositions and cells comprising these base editors.

The invention provides a fusion protein with a tribasic group editing function and application thereof, and relates to the technical field of basic group editing. The fusion protein comprises adenine deaminase, N-methylpurine DNA (deoxyribonucleic acid) glycosylase and nuclease. Aiming at the problem that an existing base editing tool cannot edit by taking three bases as substrates at the same time, the invention provides a novel three-function base editor which is named as A, Camp; and G-BE. A, Camp; the G-BE can be edited by independently using A, C and G as substrates to realize the editing activity of A-G / T / C, C-T / G / A and G-T / A / C, and can also be edited by simultaneously using A, C and G as substrates on alleles.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing proteins or protein domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing proteins or domains, are provided.

The invention discloses cytosine deaminase as well as a related biological material and application thereof, and belongs to the technical field of protein. The technical problem to be solved by the invention is to excavate natural cytidine deaminase without sequence preference and improve the base editing efficiency and range. The invention provides a 15th-143rd site protein of which the amino acid sequence is SEQ ID NO.2. The invention also provides a preparation method of the protein. The invention also provides application of the protein and related biological materials thereof in single base editing. According to the application, metagenome big data is utilized, comprehensive and systematic information mining analysis is carried out on novel cytidine deaminase, and a protein L85 which is highly similar to a known DddA protein structure but low in sequence similarity is selected from the novel cytidine deaminase for functional verification. The protein L85 has high catalytic activity as cytidine deaminase, is not limited by target sequence preference, can be widely applied to gene editing in vitro and in cells / tissues, and has good application potential and development value.

The present disclosure provides Cas9 variants, and base editors comprising these variants, that recognize non-canonical protospacer adjacent motifs (PAMs) and have less restrictive PAM requirements for editing. The present disclosure provides Cas9 protein variants comprising one or more amino acid substitutions relative to wild-type Nme2Cas9. Fusion proteins comprising the Cas protein variants described herein are also provided by the present disclosure. Further provided herein are methods for editing a target nucleic acid using the Cas variants and fusion proteins provided herein. The present disclosure also provides guide RNAs, complexes, polynucleotides, cells, kits, and pharmaceutical compositions. Further described herein are phage-assisted continuous evolution (PACE) systems, vectors, methods, and devices.

The invention discloses corynebacterium glutamicum for producing N-acetylglucosamine and application of the corynebacterium glutamicum, and belongs to the technical field of synthetic biology. Specifically, the corynebacterium glutamicum overexpresses a GNA1 gene from a brewer's yeast S288C controlled by a tacM promoter, a glmS gene from corynebacterium glutamicum S9114 and a yqab gene from escherichia coli K12 MG1655, and simultaneously has five gene defect types of nagA, nagB, ldhA, msccg and msccg2 in a genome and two gene single base mutation types of odhA and glnA, and the codon of the glnA for coding the 405th amino acid is mutated from TAC to TTC. By blocking excretion of glutamic acid of corynebacterium glutamicum and enhancing conversion of glutamic acid to glutamine, the supply capacity of a precursor of corynebacterium glutamicum is enhanced, and an optimal way for synthesizing N-acetylglucosamine is obtained, so that efficient production of N-acetylglucosamine in corynebacterium glutamicum is realized, and the yield of N-acetylglucosamine in corynebacterium glutamicum is increased. The method has important economic value and social significance.

The invention provides a molecular marker for detecting pepper light mottle virus L-series resistance genes and application of the molecular marker, and belongs to the technical field of genetic engineering, the molecular marker comprises SNP1 and SNP2, the SNP1 is a base T at the 2574th site of a yellow lantern pepper genome sequence with the NCBI login number of AB523370, and the SNP2 is a base T at the 2832nd site of the yellow lantern pepper genome sequence with the NCBI login number of AB523370; the specific gene sequence is a specific gene sequence of an L-series resistance gene L1c type gene, and the specific gene sequence is a basic group CA at the 2872 site to the 2873 site, a basic group TT at the 2877 site to the 2878 site, a basic group TG at the 2882 site to the 2883 site and a basic group AGA at the 2885 site to the 2887 site of the yellow lantern pepper with the NCBI accession number being AB523374. By utilizing the molecular marker and the specific sequence, the L-series resistance gene types of the yellow lantern pepper and the annual pepper can be rapidly and accurately detected, and the molecular marker can be used for molecular marker-assisted selective breeding.

A deaminase, a fusion protein and the use thereof. The deaminase contains an amino acid sequence having at least 50% sequence identity to any one of SEQ ID NOs: 1-6, 39-164 and 297-312, and the deaminase can deaminize at least one nucleotide in a target polynucleotide. The deaminase can be fused with a nucleic acid-binding polypeptide to obtain various base editors for the sequence-specific base conversion of the target polynucleotide. The performance of the editors can be further improved by means of fusing one or two UGIs, fusing a Gam protein and other strategies.

The invention relates to a single nucleotide polymorphism (SNP) primer of a DSTN gene related to musk ketone content in natural musk and application of the single nucleotide polymorphism (SNP) primer, and belongs to the technical field of biological detection. The invention provides a single nucleotide polymorphism (SNP) primer of a DSTN gene related to the musk ketone content in natural musk, a molecular marker corresponding to the SNP primer is located at a 682371th basic group of a chromosome 23 of a forest musk deer reference genome, the basic group is mutated into A or C, the genotypes are AA and AC, and deoxyribonucleotide sequences of the SNP primer are shown as SEQ ID NO: 1 and SEQ ID NO: 2. According to a breeding target, by eliminating A / A genotype individuals and retaining A / C genotype individuals, the method has the beneficial effects that the relative content character of musk ketone in musk of the forest musk deer is efficiently and quickly identified, and a scientific basis is provided for early breeding of high-quality forest musk deer. The detection method disclosed by the invention is simple and easy to operate, can be carried out in a laboratory, and can also be applied to a genome breeding technology.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

The invention belongs to the technical field of gene editing, and discloses a base editor system and application. According to the invention, a heterologous peptide sequence is inserted into IscB protein, the obtained peptide embedded IscB is fused with deaminase or glycosylase to construct a base editor, and DNA base editing is carried out. According to the base editor system, the size limitation is overcome, and single AAV delivery is achieved; the editing efficiency is obviously improved; the broad-spectrum applicability is realized; the system optimization is more flexible; the method has efficient editing potential in vivo, and the off-target effect is reduced. The invention provides an important new strategy for hyperlipemia treatment and provides a basis for gene therapy of other hereditary diseases.

The invention belongs to the technical field of genes, and discloses a method for efficiently expressing a foreign protein based on an NC048604-1 site in a CHO cell genome. A site for stably expressing protein in the CHO cell genome is located in the 94142000 to 94148000 basic group range of the CHO cell genome NC048604-1, and the nucleotide sequence of the site is as shown in SEQ ID NO: 1. According to the invention, different protein genes are introduced at fixed positions in a CHO cell genome, and stable expression is carried out.

Provided is an improved cytosine base editing system with reduced genome-wide off-target effects and a narrow editing window, the system comprising a base editing fusion protein comprising an APOBEC3B deaminase or an APOBEC3B deaminase mutant fused to a CRISPR effector protein.

The invention belongs to the field of gene engineering, and particularly relates to an ecdysone receptor gene LvECR and application thereof. The base sequence of the ecdysone receptor gene LvECR is as shown in SEQ ID NO: 1. The amino acid sequence of the protein encoded by the LvECR gene is as shown in SEQ ID NO: 2. The invention also discloses an application of the ecdysone receptor gene LvECR in inhibition of pathogenic vibrio infection. It is verified for the first time that the prawn ecdysone receptor gene LvECR plays an important role in prawn acute hepatopancreatic necrosis, disease occurrence can be aggravated by inhibiting expression of the coding gene of the prawn ecdysone receptor gene LvECR, disease symptoms can be reduced by injecting in-vitro recombinant protein of the factor, and the huge application potential of the prawn ecdysone receptor gene LvECR in disease prevention and control is shown.

The invention provides a polygene base editing carrier, an editing method and application, and belongs to the technical field of gene editing. According to the polygene base editing vector provided by the invention, the mode that the replicon element is connected with U6 and sgRNA in series is utilized, so that the plasmid containing the replicon element can continuously exist in a cell, and other elements on the plasmid can be continuously expressed in the cell, so that the polygene silencing efficiency is improved. The invention also provides a tandem carrier, namely an epi element in the carrier is removed. The two multi-gene editing vectors constructed in the embodiment of the invention have silence editing effects on targeting sites, and the editing efficiency of the vector containing the epi element is higher. According to the invention, a 5-gene silent homozygous cell line is obtained through a drug enrichment screening method, and a material and a method are provided for disease-resistant breeding.

A Cas12 protein and a use thereof. The amino acid sequence of the Cas12 protein comprises or is a sequence having at least 50% sequence identity to SEQ ID NO: 1, and the amino acid sequence of the Cas12 protein comprises or is a sequence different from SEQ ID NO: 1 in amino acids at one, two or more positions. The amino acid sequence of the Cas12 protein comprises or is an amino acid sequence having at least 50% identity to SEQ ID NO: 18, and a PAM sequence identified by the Cas12 protein is A. By carrying out rational and non-rational mutation on the amino acid sequence of a natural Cas12 protein, the gene editing efficiency of the natural Cas12 protein in mammalian cells is improved. The Cas12 protein achieves higher editing efficiency and / or a lower off-target rate, and the PAM sequence is a single A base, so that many target sequences which are previously difficult to edit can be edited, thereby greatly expanding the editing range.

The invention belongs to the field of variation or genetic engineering, and discloses a plant C-to-K base editor. The technical problem to be solved by the invention is how to realize C-to-K base editing in plants. The invention discloses a basic group editor for plant C to K. The basic group editor contains a Cas protein and uracil DNA glycosylase, wherein the amino acid sequence of the uracil DNA glycosylase is 20th to 244th sites of SEQ ID No.2. Experiments prove that the base editor provided by the invention realizes targeted C-to-K editing with base editing efficiency as high as 66.66%, and the range of the base editor is widened. The base editor opens up a way for optimizing crop traits (such as disease resistance, drought resistance and yield) by accurately solving mononucleotide variation affecting plant traits.

The present invention discloses a recombinant glycosylase base editing system and its application. The recombinant glycosylase base editing system of the present invention is an APOBEC-nCas9-Ung1 or APOBEC(R33A)-nCas9-Rad51-Ung1 base editing system; the base editing system APOBEC-nCas9-Ung1 includes cytosine deaminase APOBEC, nuclease nCas9 and uracil DNA glycosylase Ung1; the base editing system APOBEC(R33A)-nCas9-Rad51-Ung1 includes cytosine deaminase APOBEC(R33A), nuclease nCas9, single-stranded DNA binding protein Rad51 and uracil DNA glycosylase Ung1. Experiments have shown that the recombinant glycosylase base editing system of the present invention can improve the editing efficiency of base C to base G in the genome sequence of an organism or a biological cell and the purity of the editing product.

The invention discloses a method for identifying duck palm weight and related molecular marker application thereof, and relates to the technical field of molecular marker-assisted selection, a primer pair is used for amplifying a DNA fragment containing a 63823029th base polymorphic site from the 5'terminal on a fourth chromosome of a duck reference genome IASAASPekinDuckT2T, and the primer pair is used for amplifying a DNA fragment containing the 63823029th base polymorphic site from the 5 '-terminal on the fourth chromosome of the duck reference genome IASAASPekinDuckT2T. The duck reference genome IAASAASPekinDuckT2T is a duck reference genome sequence in a GenBank database, and the duck reference genome sequence is a duck reference genome sequence in the GenBank database; the duck is a Chinese and new white feather meat duck, and the primer pair consists of a DNA (deoxyribonucleic acid) molecule as shown in SEQ ID No.2 and a DNA molecule as shown in SEQ ID No.3. According to the method for identifying the duck foot weight and the application of the related molecular marker, the genotype of the duck can be judged by detecting the genome DNA of the duck, early living screening of the duck foot weight character is achieved, phenotype determination after slaughtering is not needed, the breeding period is remarkably shortened, and the breeding cost is reduced.

The invention relates to the field of gene engineering. In particular, the present invention relates to cytosine deaminases and their use in base editing. More specifically, the invention relates to a method for screening and identifying a deaminase, a base editing system based on a newly identified cytosine deaminase, a method for editing a target sequence in a genome of an organism (such as a plant) by using the base editing system, and a method for screening and identifying the target sequence. As well as genetically modified organisms (e.g., plants) and progeny thereof produced by the method.

The application discloses a SNP molecular marker for identifying the genetic sex of Procambarus clarkii, wherein the nucleotide sequence of the SNP molecular marker is shown as SEQ ID No. 1, and a male individual is shown as GG homozygote at the 43th base position of the nucleotide sequence of the SNP molecular marker, and a female individual is shown as GA heterozygote at the 43th base position of the nucleotide sequence of the SNP marker. The application further discloses primers and kits for detecting the SNP molecular marker, and application of the reagents, primers and kits for detecting the SNP molecular marker in genetic sex identification of the Procambarus clarkii. The SNP marker is closely related to the genetic sex, and can be used for conveniently and quickly identifying the genetic sex of the Procambarus clarkii in the process of family breeding and unisexual breeding of the Procambarus clarkii, and provides a theoretical basis for identification of sex-reversed individuals in the breeding process.

The invention discloses application of a fusion protein KSa-SspCas9 and a mutant thereof in gene editing, and belongs to the field of genetic engineering. SspCas9 protein in Staphyloccus sp.17KM0847 bacteria is excavated through analysis of bioinformatics, the protein has PAM recognition capability of NNG, a PI structural domain of the SspCas9 protein is fused with a non-PI structural domain of the SaCas9 protein to obtain the fusion protein KSa-SspCas9, and the fusion protein KSa-SspCas9 is used for gene editing. The fusion protein has higher base editing efficiency. Meanwhile, the specific site of the fusion protein is mutated, and the PAM recognition range is greatly widened through PAM sequences of NRN and NYN which can be recognized by mutants. Through discovery of the Cas9 protein and the gene editing system, the variety of gene editing tools is expanded, and the Cas9 protein and the gene editing system have wide application prospects in the aspects of disease treatment, animal model preparation, plant breeding and the like.

The invention belongs to the field of variation or genetic engineering, and discloses a T-to-G base editor for plants. The technical problem to be solved by the invention is how to realize T-to-G base editing in plants. The base editor for the plant from T to G disclosed by the invention contains a Cas protein and uracil DNA glycosylase, wherein the amino acid sequence of the uracil DNA glycosylase is 20th to 244th sites of SEQ ID No.2. The invention also discloses a preparation method of the base editor for the plant from T to G. Experiments prove that the base editor provided by the invention realizes targeted T-to-G editing with base editing efficiency as high as 78.05%, not only can solve the limitation that T direct editing cannot be realized in a rice genome, but also can induce exon jumping to obtain a required mature transcript. The base editor provided by the invention widens the targeting range of the base editor, and can also be applied to expression regulation of genes.

A composition of matter includes an antisense phosphorodiamidate morpholino oligomer (MO) that includes an MO base sequence. The MO base sequence is arranged to bind a corresponding complementary base sequence of messenger RNA (mRNA) transcribed from one or more genes for the beta subunit of human chorionic gonadotropin (hCG-β). An inventive method, for suppressing intracellular synthesis of a beta subunit of human chorionic gonadotropin (hCG-β), includes introducing such an MO into one or more cells.