667 results about "Insertion" patented technology

The invention belongs to the technical field of gene sequencing, and particularly relates to a method for efficiently identifying an exogenous DNA insertion site in a genome by using a high-throughput sequencing technology. The method comprises the following steps: (a) extracting a sample, building a database and sequencing; (b) data quality control; (c) comparing data; (d) filtering comparison results; (e) preliminary screening; and (f) screening and identifying the positions of the exogenous insertion elements. According to the method, the high-throughput sequencing data and the genome and exogenous fragment sequences are compared and analyzed to obtain the candidate positions of the insertion sites, so that the test efficiency can be remarkably improved, manual judgment and screening work can be effectively replaced, and the result accuracy can be improved.

Systems and methods for targeted gene modification, targeted insertion, perturbation of gene transcripts, and nucleic acid editing. Novel nucleic acid targeting systems comprise components of CRISPR systems and non-LTR retrotransposon elements.

The invention relates to a molecular marker closely linked with a pepper fruit length regulation gene CaFL1 and application of the molecular marker. The molecular marker is insertion / deletion of three basic groups GAC at 223149123-223149125th positions of chromosome 10 of a pepper genome, and the position of the molecular marker in the genome is determined according to a Qieme reference genome. Specifically, when the genotype of the molecular marker is InGAC: InGAC, the pepper has a long fruit phenotype, when the genotype is InGAC: DelGAC, the pepper has an intermediate phenotype, and when the genotype is DelGAC: DelGAC, the pepper has a short fruit phenotype. Through verification, the accuracy rate of the molecular marker reaches 100%, early, massive and accurate prediction and directional improvement of the pepper fruit length can be realized, and a new way is provided for screening and identifying the pepper fruit length and breeding the pepper fruit length.

The invention relates to the technical field of crop molecular genetic markers, and discloses an InDel molecular marker of avena nuda and application thereof.The molecular marker is a group of 314bp deletion variation and 2bp insertion variation in the 610439484-610440026 interval of the 5C chromosome position of a reference genome of an avena nuda variety OT3098, the nucleotide sequence of the deletion variation is shown as SEQ ID NO.1, the nucleotide sequence of the insertion variation is shown as SEQ ID NO.2, and the nucleotide sequence of the insertion variation is shown as SEQ ID NO.3. The nucleotide sequence of the insertion variation is 5 '-AA-3'. The molecular marker provided by the invention can realize stable detection in agarose gel electrophoresis by combining a specific primer pair (SEQ ID NO.2-3) with an optimized touchdown PCR (Polymerase Chain Reaction) program, has the advantage of being not influenced by a tissue part and a growth period, and provides an efficient and reliable technical means for oat germplasm identification, seed purity detection and molecular breeding.

The present disclosure provides compositions and methods for the targeted modification of RNA molecules by RNA prime editing. The compositions and methods may be conducted invitro or in vivo within cells (e.g., human cells) for the therapeutic correction of disease-causing mutations and / or installation of motifs or mutations in RNA molecules of interest as a tool for scientific research. The disclosure provides compositions and methods for conducting RNA prime editing of a target RNA molecule (e.g., an RNA transcript) that enables the incorporation of one or more nucleotide changes and / or targeted mutagenesis of a target RNA molecule. The nucleotide change can include a single-nucleotide change, an insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides a variety of configurations of the RNA prime editors each comprising a nucleic acid programmable RNA binding proteins (napRNAbp), such as Cas13, and an RNA-dependent RNA polymerase (RDRP), which are provided as fusion proteins or which can be separately provided in trans. The RNA prime editors are guided to a target RNA site by a guide RNA, which can be a rpegRNA that includes a template region for the synthesis of an RNA sequence to be installed on the RNA molecule attached to an available 3′ terminus. In others embodiments, the RNA template can be provided in trans.

The invention discloses a molecular marker related to thousand seed weight of wheat, a detection primer and application of the molecular marker and the detection primer, and belongs to the technical field of molecular marker-assisted breeding. The nucleotide sequence of the molecular marker TaDTX50-STS is as shown in SEQ ID NO. 1; basic groups from the 502 site to the 661 site of the sequence as shown in SEQ ID NO. 1 have insertion / deletion mutation. The InDel molecular marker related to the thousand seed weight character in a wheat genome is identified and named as TaDTX50-STS, and by detecting the polymorphism or genotype of the InDel molecular marker, the InDel molecular marker can be used for identifying or assisting in identifying the thousand seed weight character of wheat, assisting in breeding dominant varieties with high thousand seed weight of wheat and improving the wheat breeding efficiency.

The invention discloses application of Indel marker dosage typing in detection of sweet potato soft rot resistance, and belongs to the technical field of plant genetic engineering. The Indel molecular marker related to the sweet potato soft rot resistance is obtained through screening, the nucleotide sequence of the Indel molecular marker is shown as SEQ ID NO.2, and an 8bp sequence is inserted into or deleted from the 161st site of the sequence shown as SEQ ID NO.2. Experiments find that target bands of amplification products are obtained through amplification of the Indel molecular marker and electrophoresis, dosage values of two genotypes are obtained according to gray values of the dyed electrophoretic bands, and results show that the insertion type Indel molecular marker is positively correlated with the sweet potato soft rot resistance. The method is of great significance to the dose effect research and molecular assisted breeding of sweet potato gene functions.

The invention belongs to the technical field of gene editing, and particularly relates to optimized omega RNA and gene editing application thereof. According to the optimized omega RNA, base mutation is carried out on the basis of wild type omega RNA with a gene sequence shown as SEQ ID NO.1, and by taking the 5'end of the wild type omega RNA, the 36th paired base UA is replaced with GC, the 37th paired base UA is replaced with AU, the 38th paired base UG is replaced with AU, the 50th paired base UG is replaced with CG, and the 51st paired base UA is replaced with GC. Compared with wild type omega RNA, the omega RNA has the advantages that the maximum enhancement multiple of the insertion and deletion activity is 16.7 times, 15 pairs of paired bases from the 40th site to the 54th site of the omega RNA are further deleted, and the editing efficiency is further improved.

The invention belongs to the technical field of gene engineering, and particularly relates to a specific sequence of transgenic soybean medium yellow 6106 and application of the specific sequence, the specific sequence comprises an exogenous insertion element complete sequence and a flanking sequence of the exogenous insertion element complete sequence, and the nucleotide sequence of the specific sequence is shown as SEQ ID NO.1. The specific detection method is established through the specific sequence of the transgenic soybean yellow 6106, whether a sample to be detected contains the component of the transgenic soybean yellow 6106 or not can be effectively detected, and the detection method is rapid, simple, convenient, accurate and reliable.

The invention provides a genome editor enDelIscB and a genome editor enDelIscB-T5E based on an IscB system, and further provides a base editor ICBE and a base editor IABE based on the enDelIscBD60A. According to the invention, a genome editor and a base editor which are more compact in size and higher in cleavage activity and insertion / deletion efficiency are realized by optimizing a single mutation site of the activity of a DelIscB protein cleavage plasmid, an optimized variant of the DelIscB protein and an optimized sgRNA variant of the DelIscB. According to the present invention, it is verified that the albinism model is efficiently constructed by using the enDelIscB editor and the enDelIscB-T5E editor through micro-injection of the mouse embryo, such that the enDelIscB editor and the enDelIscB-T5E editor have great potential in the field of genome editing.

The invention provides a cotton transformation event cD46-2 as well as a primer pair and a method for detecting the cotton transformation event cD46-2. The cotton plant carrying the cD46-2 transformation event comprises the event, namely the joint of an exogenous insertion DNA sequence and a cotton genome DNA sequence, on a group A chromosome 2. A detection primer can be designed by using an exogenous insertion DNA sequence and a DNA sequence of a junction region on a flanking cotton genome, and is used for specific detection of a cD46-2 event. The detection method aiming at the cD46-2 event can provide a convenient means for tracking the specific gene insertion event for the application of breeding by using the plant event, and the specific gene insertion event can be used as a molecular marker to improve the working efficiency of fiber improvement breeding by using the transformation event.

The invention discloses enterobacter mori capable of resisting fish aeromonas and application of the enterobacter mori, the bacterial strain is classified and named as enterobacter mori SCH0241, the enterobacter mori SCH0241 is preserved in the China Center for Type Culture Collection on October 17, 2024, and the preservation number is CCTCC NO: M 20242236. The enterobacter mori SCH0241 disclosed by the invention is derived from the intestinal tract of high-stress-resistance healthy hybrid crucian carassius auratus No. 2, has good contact dependence antibacterial activity on aeromonas veronii, and can improve the capability of resisting aeromonas veronii infection of zebra fish. Through construction of an insertion mutation library and phenotype screening, it is found that a gene cluster of SCH0241, which exerts contact inhibition toxicity, is T6SS-2. The enterobacter mori SCH0241 disclosed by the invention provides a new powerful tool for biological prevention and treatment of fish diseases caused by aeromonas veronii.

Among other things, provided herein are systems that replace the natural random integration activity of a retrovirus with site-specific integration machinery. This approach allows for a more precise targeting of a gene of interest into a human genome, e.g., for therapeutic purposes. The system may include integration-deficient retrovirus (e.g., lentivirus) (IDLV), in which the natural integration activity has been reduced (e.g., by mutation to the viral integrase polypeptide). Instead, the system may comprise a site-specific recombinase (e.g., a serine recombinase, e.g., a serine integrase) capable of directing insertion of a template DNA, or portion thereof, into a desired site in the human genome.

The invention relates to an anti-human HHLA2 nano antibody and application thereof, a variable region of the nano antibody comprises CDR1, CDR2 and CDR3, an amino acid sequence of the CDR1 comprises any one of sequences as shown in SEQ ID NO: 29-36 or a mutation thereof, an amino acid sequence of the CDR2 comprises any one of sequences as shown in SEQ ID NO: 37-46 or a mutation thereof, an amino acid sequence of the CDR3 comprises any one of sequences as shown in SEQ ID NO: 47-56 or a mutation thereof, and the variable region of the nano antibody comprises CDR1, CDR2 and CDR3. The mutation is the substitution, insertion or deletion of 1, 2, 3 or 4 amino acids in the amino acid sequence. The nano antibody disclosed by the invention can be specifically combined with tumor cells expressing human HHLA2, and the killing effect of immune cells on the tumor cells can be improved by blocking a signal channel between the HHLA2 and KIR3DL3.

The invention discloses a method for site-specific replacement or insertion of DNA fragments in a plant genome, and belongs to the technical field of biology. The method disclosed by the invention comprises the following steps: editing a genome of a plant protoplast by adopting a CRISPR / Cas9 system and taking a double-stranded DNA repair template as a donor template to obtain an edited protoplast; and carrying out culture regeneration on the edited protoplast by utilizing a plant protoplast transformation method to obtain a site-directed mutagenesis plant in the genome so as to realize site-directed mutagenesis of the target plant genome. The method disclosed by the invention can be used for accurately replacing DNA in a plant genome at a fixed point. According to the invention, a recognition sequence of recombinase can be accurately inserted into a genome at a fixed point through homologous recombination, and then accurate fixed-point replacement of a gene large fragment is realized by virtue of a specific recombinase system.

Compositions and methods comprising a deaminase for targeted editing of nucleic acids are provided. Also provided are compositions and methods for localizing a heterologous polypeptide to a target DNA molecule, and compositions and methods for targeted editing of nucleic acids. Fusion proteins comprising an RNA-guided nuclease (RGN) and at least one heterologous polypeptide inserted therein are provided, as well as fusion proteins comprising a DNA binding polypeptide and a deaminase. The heterologous polypeptide may be a pilot editing polypeptide or a base editing polypeptide. Compositions also include nucleic acid molecules encoding a deaminase or fusion protein. Vectors and host cells comprising the nucleic acid molecules encoding the deaminase or fusion protein are also provided.

Provided is a heat-resistant D-allulose-3-epimerase. A polypeptide shown in any one of the following (1) to (3):(1) a polypeptide consisting of an amino acid sequence having one or two or more amino acid substitutions selected from the group consisting of A56E, N57H, R58G and F111W in SEQ ID NO:1;(2) a polypeptide consisting of the amino acid sequence having substitution, addition, insertion or deletion of one or several amino acid residues in the amino acid sequence described in (1) above, having D-allulose-3-epimerase activity and retaining one or two or more amino acid substitutions selected from the group consisting of A56E, N57H, R58G, and F111W; and(3) a polypeptide having a 90% or more sequence identity to the amino acid sequence described in (1) above, having D-allulose-3-epimerase activity and retaining one or two or more amino acid substitutions selected from the group consisting of A56E, N57H, R58G, and F111W.

Described herein are methods of preparing an enriched library of nucleic acids, comprising: (a) identifying a patient-specific panel of somatic variants present in a tumor sample from a patient, (b) generating a subset panel of somatic variants from the patient-specific panel, wherein the subset of somatic variants comprises one or more of: multi -nucleotide variants, insertions and deletions, and genomic rearrangements; (c) preparing a sample of cell-free DNA (cfDNA) fragments from the patient for sequencing; and (d) selectively enriching the cfDNA for the subset of somatic variants to generate an enriched library.

The invention discloses a muskmelon CmACS11 gene promoter structure variation SV352 and application thereof in regulation and control of female development of muskmelons, and relates to the technical field of biology. It is found for the first time that insertion of 352bp exists on the CmACS11 promoter, the CmACS11 promoter is named as SV352, the nucleotide sequence of the CmACS11 promoter is shown as SEQ ID No: 3, and the CmACS11 promoter is closely linked with female complete development characters of muskmelons and has dominant inheritance. Furthermore, through in-situ hybridization and transgenosis verification, the SV352 causes the loss of selective expression characteristics of the CmACS11, so that the CmACS11 is expressed in all flower buds, and the expression is advanced, which is a key variation for controlling complete development of female organs of muskmelons. The SV352 variation provides a new variation resource for creating a muskmelon female line breeding material.

The invention discloses a DNA sequence reconstruction method and system based on multi-scale attention and contrast learning, and relates to the technical field of DNA storage data reconstruction. Comprising the following steps: collecting a plurality of DNA sequence copies, screening out abnormal length sequences, and constructing a standardized clustering data set; performing one-hot coding and filling processing on the DNA sequence; extracting context dependent features and cross-sequence variation features; an Inter-Sequence multi-head attention mechanism is constructed to calculate the similarity between the sequences, and a weighted sequence tensor is generated; a global dependency relationship in the sequence is extracted through an Intra-Sequence multi-head attention mechanism; local offset features caused by insertion and deletion errors are extracted through a multi-size convolutional network; inputting a double-layer long-short-term memory network for sequence-level modeling, and outputting base reconstruction probability distribution; and constructing positive and negative sample pairs, calculating comparison loss, combining cross entropy loss to form a joint loss function, and outputting a high-precision DNA sequence reconstruction result. The method has high accuracy and robustness under the conditions of complex noise and multiple types of errors.

The invention discloses an Indel marker related to a corn kernel protein content character and application of the Indel marker, and belongs to the technical field of plant molecular genetics and crop molecular design breeding. The Indel marker is located on a gdh2 gene of a chromosome 10 of a corn genome, and insertion or deletion of a nucleotide sequence as shown in SEQ ID NO.5 occurs in an intron region between a second exon and a third exon. By applying the molecular marker-assisted selection technology, high-protein new germplasms S3317 (the crude protein content is 17.0%) and JS231148 (the crude protein content is 15.6%) have been successfully cultivated, the increase amplitudes of the high-protein new germplasms and JS231148 are both more than 15% compared with those of the conventional control, and the lysine content reaches 0.41% and reaches the national high-quality protein corn standard.

The invention belongs to the technical field of molecular markers, and discloses an alfalfa yield identification method, a molecular marker and application, a nucleotide sequence of the molecular marker is shown as SEQ ID NO.4 or SEQ ID NO.5, an upstream primer sequence of a primer pair for amplifying the molecular marker is shown as SEQ ID NO.1, and a downstream primer sequence of the primer pair is shown as SEQ ID NO.2. The invention further discloses a method for identifying the yield of alfalfa. The molecular marker is located on a chromosome 5 of medicago sativa, and the nucleotide sequence of an insertion or deletion fragment of the molecular marker is shown as SEQ ID NO.3 in a sequence table. The InDel molecular marker and the primer pair provided by the invention can be used for predicting, identifying or assisting in identifying the yield traits of medicago sativa. Meanwhile, the invention further provides a method for identifying the alfalfa yield character by using the InDel molecular marker, and the method is simple, convenient and rapid, accurate in identification result and good in popularization and application prospect.

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 belongs to the technical field of microbial engineering, and discloses a method for producing eicosapentaenoic acid (EPA) with high yield by schizochytrium limacinum based on random mutation and low-temperature screening and application, and the method comprises the following steps: driving overexpression of a G418 resistance gene through a P2520 promoter, constructing a random insertion mutant library, and screening to obtain a schizochytrium limacinum mutant strain with a randomly mutated genome; coating in a culture medium containing G418 resistance for culturing, and screening out strains which are resistant to low temperature and good in growth state at 15 DEG C; and inoculating into a fermentation culture medium, adding MgSO4 with the final mass concentration of 0.1% at the initial stage of fermentation, supplementing MnCl2 with the final mass concentration of 0.05% when fermentation is performed for 72 hours, and culturing to obtain a mutant strain. According to the invention, the G418 resistance gene carried by the plasmid is randomly inserted into the schizochytrium limacinum genome, and low-temperature induced screening is combined, so that the target limitation of traditional homologous recombination is broken through, and a stable strain with multiple mutation superposition can be obtained through enrichment.

The invention belongs to the technical field of cellular immunity, and relates to a non-virus-dependent engineering induced pluripotent stem cell as well as a preparation method and application thereof. A coding gene of a chimeric antigen receptor is inserted into an SIRPA gene on a genome of the engineered induced pluripotent stem cell. According to the invention, iPSC cells are edited in a SIRPA gene position fixed-point insertion mode, simultaneous knockout of SIRPA and integration of an exogenous chimeric antigen receptor CAR are realized, and CAR-iMac can be further induced and differentiated, so that CAR-iMac is differentiated to mononuclear / macrophage tumor targeted cells, the phagocytic ability of CAR-iMac is enhanced, tumor antigens are further presented, the tumor immune microenvironment is improved, and the anti-tumor immunity is enhanced; the method effectively solves the corresponding problem of directly extracting the natural macrophages from the human body, reduces the production cost and the time cost, is beneficial to clinical application, and in addition, utilizes a non-virus-mediated gene editing means to remarkably improve the genetic silencing phenomenon.

The invention belongs to the field of gene engineering, and particularly relates to a novel recombination system in aeromonas and application of the novel recombination system. A novel recombination system in the aeromonas disclosed by the invention contains a homologous recombination system expression vector, the expression vector expresses an operon under the control of an inducible promoter, and the operon contains three genes of a coding host specific exonuclease RedAahy, a single-stranded DNA annealing protein RedBahy and a single-stranded DNA binding protein RedSahy; the operon is derived from an aeromonas hydrophila (Aeromonas hydrophila) plasmid pR148, and is characterized in that the operon is derived from the aeromonas hydrophila (Aeromonas hydrophila) plasmid pR148. By utilizing the recombination system, efficient, simple and convenient gene insertion and deletion of aeromonas are successfully realized, and the application prospect is wide.

We describe compositions of DNA donor molecules compatible with various gene insertion methods with major advantages for delivery, nuclear localization, and immune evasion. The modified DNA donor molecules are largely single stranded but contain short regions of double-strand DNA to reconstitute enzyme binding sites, and can be combined with other approaches to enhance donor recruitment into the nucleus.

Provided is an efficient and precise plant gene knockout method. First, a sequence containing a stop codon cluster is inserted into a genome by means of dual pegRNA to obtain, by means of screening, a stop codon cluster sequence capable of efficiently and precisely knocking out a target gene. A prime editing system and multiple pegRNA programming can be used in combination with the method to enable various genomic modifications such as efficient and precise knockout of one or more target genes, base substitutions, and small fragment insertions, deletions, and substitutions with just one prime editing protein.

The invention belongs to the technical field of crop molecular markers, and particularly discloses a molecular marker for identifying the salt tolerance of brassica napus in the germination period and application of the molecular marker. The molecular marker is located at the position of an A02 chromosome scafoldA028707654 of a brassica napus reference genome ZS11.v10, and the allelotype of the molecular marker is wild type C or mutant type CATATT insertion. The invention also provides a specific primer group for detecting the molecular marker, and the salt tolerance (sensitive type or tolerance type) of the brassica napus line can be quickly identified according to the existence of 411bp and 416bp bands through double PCR amplification and electrophoresis detection. The molecular marker is significantly associated with salt tolerance phenotypes, the detection method is rapid, accurate and low in cost, the molecular marker can be used for brassica napus salt tolerance germplasm resource screening and molecular marker-assisted breeding, and the breeding efficiency is significantly improved.

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.