57results about How to "High target shooting efficiency" patented technology

The invention discloses a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 system capable of simultaneously knocking out KRAS genes and EGFR genes. The system comprises sgRNA for specifically targeting KRAS genes and sgRNA for specifically targeting EGFR genes, wherein a corresponding DNA sequence of the sgRNA for specifically targeting KRAS genes is shown as SEQ ID NO.1 or / and SEQ ID NO.2; and a corresponding DNA sequence of the sgRNA for specifically targeting EGFR genes is shown as SEQ ID NO.11 or / and SEQ ID NO.12. The invention further discloses application of the system in preparation of medicines for treating cancers. The CRISPR-Cas9 system disclosed by the invention is capable of simultaneously and efficiently knocking out two cancer driving factors KRAS and EGFR which are highly-expressed in lung cancer. The system is simple in operation and high in knockout efficiency and is expected to be applied to treatment of the lung cancer. The system disclosed by the invention is applicable to multiple cancers with abnormal expressions of the EGFR and KRAS.

The invention discloses a plant gene knockout vector based on a CRISPR / Cas9 technology. The monocotyledon plant gene knockout vector is characterized in that the plant gene knockout vector is a monocotyledon plant CRISPR / Cas9 knockout vector pCambin1300-OsU3-Cas9, and contains the AarI insertion site. According to the present invention, with the CRISPR / Cas9 plant knockout double-component vector pCambin1300-OsU3-2x35s-Cas9 formed from a sgRNA-OsU3 structure sequence comprising the AarI insertion site and a 2x35s-Cas9-ter sequence, the purpose of the efficient and rapid construction of the monocotyledon plant gene knockout vector can be achieved through the one-step digestion linkage method; and the provided CRISPR / Cas9 knockout vector pCambin1300-OsU3-2x35s-Cas9 has characteristics of high targeting efficiency and extremely low undershoot efficiency, and can be used for plant target gene knockout or can be used for plant gene editing in a kit form.

The invention belongs to the field of fish molecular breeding, and particularly relates to a fish breeding method for improving CRISPR-Cas9 gene editing and passage efficiency through fish roe preserving fluid. According to the method, the fish roe preserving fluid technology, the microinjection technology an the CRISPR-Cas9 gene editing technology are ingeniously combined, the targeting efficiency of the CRISPR-Cas9 gene editing technology and gene editing passage efficiency are greatly improved, the screening time needed by a gene editing method for fish breeding is obviously saved, and the method is of great significance in promoting rapid development of gene editing fish breeding.

The invention relates to a moveable reflector laser collimator, a moveable reflector target surface sensor and a laser collimating method thereof. The moveable reflector laser collimator comprises an image acquisition device, a moveable reflector and a lens, wherein the image acquisition device is used for acquiring incident rays to form an image; the moveable reflector is used for reflecting thelaser onto the image acquisition device to form a light spot; the lens is used for transmitting a target to the image acquisition device; and the moveable reflector can move linearly relative to the image acquisition device. By providing a moveable reflector system, the small-range high-precision translation of the moveable reflector is implemented, so that the target can be quickly and accurately collimated by the laser, thereby solving the phenomenon of light spot aliasing, saving the collimating time and enhancing the targeting precision.

The invention discloses a backbone plasmid vector. In the backbone plasmid vector, a guide RNA (Ribonucleic Acid) expression cassette and a Cas9 nuclease expression cassette are located in the same binary vector; the guide RNA expression cassette is sequentially composed of a wheat TaU3p promoter, a spectinomycin resistance gene, an artificially-synthesized sgRNA framework sequence and a Poly-T terminator; the Cas9 nuclease expression cassette is sequentially composed of a ZmUBI promoter, a plant preferred codon modified Cas9 coding sequence and a 35s terminator. The invention also provides a target-sequence-contained recombinant vector constructed by using the backbone plasmid vector and used for crop gene targeting.

The present invention provides a gene knockout vector, a method for preparing a CD13 knockout porcine fibroblast or gene-edited pig, and the CD13 knockout porcine fibroblast prepared by the method. The gene knockout vector includes a gene editing vector backbone and a DNA segment linked to the vector backbone, and the vector backbone is selected from CRISPR / Cas9, CRISPR / Cas9n, CRISPR / Cpf1 or CRISPR / C2c2. The nucleotide sequence of the DNA segment is as shown in any one of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO: 3, preferably as shown in the SEQ ID NO:1. CD13 gene can be rapidly and accuratelyknocked out by the gene knockout vector and the method to obtain the CD13 gene homozygous knockout porcine fibroblast and gene-edited pig without an exogenous marker gene, a research platform is provided for porcine epidemic diarrhea, and the CD13 gene homozygous knockout porcine fibroblast and gene-edited pig have very high breeding value for disease resistance.

The invention discloses a novel method for efficiently knocking out low-transfection-efficiency cells on the basis of lentivirus expression CRISPR / Cas9. The method is characterized in that the method relates to two lentiviruses which include a lentivirus for expressing sgRNA and Cas9 and a lentivirus for expressing Cre protein; the multigene sgRNA and Cas9 are simultaneously cloned to a position between two same-direction loxp of a lentivirus vector, simultaneous multigene knockout in the low-transfection-efficiency cells is achieved, the Cas9 and sgRNA integrated in a genome are effectively removed by using the lentivirus expressing Cre, and an off-target effect caused by Cas9 and sgRNA overexpressing is prevented. The method has the advantages that the method combines the features that the lentiviruses can effective infect cleavage and non-cleavage cells and an loxp-Cre system can perform efficient removing reaction, and the system is low in toxicity, high in gene knockout efficiency, high in accuracy, short in cycle and significant to the realizing of multigene knockout in a low-transfection-efficiency cell line.

The invention discloses a sgRNA fragment. The DNA sequence of the sgRNA fragment comprises a length of a fixed sequence and a length of a DNA recognition sequence. The sgRNA can recognize different target sites on double strands of a target gene, bond with nuclease and guide nuclease to bond with the target sites of the target gene through recognition of PAM sequences at the target sites and to start random shearing; then nuclease falls off from a shearing opening, thereby forming a DSB gap; then cells repair the double strands of the target gene through a non-homologous end joining repair mechanism, which leads to frameshift mutation; and finally, the target gene is knocked out. The invention further discloses a gene fixed-point reconstruction method and a gene targeting kit. The invention elaborates a series of sgRNAs applicable to the mouse Myod1 gene, and the sgRNAs have high targeting efficiency, short construction time and low cost and can be highly efficiently used for mouse Myod1 gene targeting.

The invention relates to the field of genetic engineering and discloses a pair of polypeptides and encoding genes thereof, with sequences shown as SEQ ID NO. 1 to 4. The pair of polypeptides is used for establishing a pair of a recombinant transcription activator like effector (TALE) and a transcription activator like effector nuclease (TALEN); the TALEN is a fusion protein formed by fusing a pair of DNA recognizing proteins with a DNA severing protein respectively; and the TALEN can perform targeted severing on a target site of a human insulin gene so as to achieve targeted modification on the human insulin gene, with the characteristics of strong specificity, high efficiency and high accuracy.

The invention provides a pair of TALENs for efficiently editing a rice WAXY gene, and an identification targeting site and an application thereof, and concretely provides a transcription activator-like effect nuclease (TALEN) gene for efficiently targeting the rice endogenous WAXY gene, and a method using a recombinant plasmid containing the gene to carry out oriented targeting. The TALENs contain a pair of TALE proteins and DNA endonuclease catalytic subunits respectively fused with the TALE proteins, and can respectively identify and cut two adjacent sites of the rice endogenous WAXY gene. Synthesis of a nucleotide sequence for encoding the TALENs and construction of a vector including the nucleotide sequence are realized on the basis of designing the amino acid sequence of the TALEs, and the cell targeting efficiency is greatly improved through plasmid transfection of cells by the TALENs.

The invention discloses a pair of transcriptional activator-like effector nucleases (TALEN) as well as an encoding gene and application thereof. The pair of TALENs is obtained by fusing a pair of DNA recognition proteins with one Fok1 nuclease monomer respectively and can specifically recognize two adjacent sites on exon 6 of a human Pax6 gene and carry out digestion. When the pair of TALENs are transferred into host cells at the same time, exon 6 sites of Pax6 genes of the host cells can be targeted, and gene mutation is carried out on the targeted sites, so that targeting modification on the human Pax6 gene is realized, and the advantages of strong specificity, high targeting efficiency and high accuracy are realized.

The invention discloses a method for increasing HR (Homologous Recombination) repairing frequency of ovine embryo fibroblasts after gene editing. The method provides application of a substance for restraining the expression of an Lig4 gene in in-vitro ovine embryo fibroblasts containing gene editing DNA (Deoxyribose Nucleic Acid) fragments in preparation of products for promoting HR repairing of the in-vitro ovine embryo fibroblasts containing the gene editing DNA fragments. An experiment of the invention shows that siRNA (Small Interfering Ribose nucleic Acid) which restrains the expression of the ovine Lig4 gene is screened, the expression of the ovine Lig4 gene is restrained through the siRNA, an intracellular HR repairing path is stimulated by restraining an NHEJ (Non-homologous End Joining) repairing path of the ovine embryo fibroblasts, the frequency of adopting HR repairing by cells is increased, and a basis is provided for increasing gene targeting efficiency of the ovine embryo fibroblasts and researching accurate editing of an ovine genome.

The invention discloses a method for improving resistance of rice to HPPD inhibitor herbicides through gene editing and a special sgRNA used therein. The coding sequence of the specific sgRNA for editing a rice OsHPPD gene based on CRISPR / Cas9 in the invention is as shown in SEQ ID NO. 6. A CRISPR / Cas9 vector aiming at the rice OsHPPD gene contains a coding gene sequence for coding the specific sgRNA. A method for improving the resistance of rice to the HPPD inhibitor herbicides through gene editing comprises the following step: introducing the coding gene of the specific sgRNA and the coding gene of a Cas9 protein into starting rice to obtain transgenic rice with a mutated OsHPPD gene. Compared with the starting rice, the resistance of the transgenic rice to the HPPD inhibitor herbicides is improved.

The invention discloses a method for acquiring gene editing sheep by RNA-mediated specific FGF5 gene knockout and special sgRNA for the method. The sgRNA provided by the invention can realize specific targeted modification of sheep FGF5 gene, is RNA shown in the second to the 21st nucleotides from the sequence 4 to 5' end tail of a sequence table or RNA with the second to the 21st nucleotides from the sequence 4 to 5' tail end of the sequence table. The invention provides an effectively technical tool for sheep functional gene researches.

The invention discloses a TALEN-mediated vector for knocking out goat BLG through gene targeting and a recombinant cell. A TALEN eukaryotic expression vector aiming at a first exon of a goat beta-lactoglobulin gene and a BLG gene knockout vector containing short homologous arms are designed and constructed according to the goat beta-lactoglobulin gene and are jointly transfect goat fetal fibroblast together with mRNAs obtained by in vitro transcription based on TALEN expression vector as a template, and a BLG gene knockout cell is obtained after drug screening and identification. According to the invention, the gene targeting efficiency is greatly improved by utilizing a TALEN technology; meanwhile, the utilization of the short homologous arms facilitates more convenient and more efficient screening of the targeted cells; random integration of the TALEN expression vector in genomes is avoided by utilizing mRNAs.

The invention discloses a double-sided precise targeting device and method for an HDI plate, and relates to the technical field of double-sided targeting of the HDI plate, the double-sided precise targeting device comprises a workbench, a portal frame fixedly arranged on the workbench, and a limiting plate fixedly arranged on the top surface of a positioning table and located on the front edge of the positioning table; jacking air cylinders fixedly arranged on the positioning table are arranged under the two guide holes, and floating columns slidably installed in the guide holes are fixedly arranged at the acting ends of piston rods of the jacking air cylinders. An automatic feeding device located on the rear side of the positioning table is arranged on the workbench, and material lifting and conveying devices are arranged on the cross beam of the portal frame and located on the left side and the right side of the positioning table. The device has the beneficial effects that the working intensity of workers is relieved, the targeting efficiency of the HDI plate is greatly improved, and the drilling efficiency of the HDI plate is greatly improved.

The invention discloses a method for genetic targeting of stem spermatogonium of poultry. By using an adenovirus of poultry as a targeting vector, the method performs targeting operation on the stem spermatogonium of the poultry; the obtained genetically modified stem spermatogonium can be used for transplanting testis and obtaining a transgenetic gene; and by mating the normal poultry, the stem spermatogonium can be further used for breeding the transgenetic poultry. The method has the advantages that: the method can effectively realize the high-efficiency genetic targeting of the stem spermatogonium of the poultry; the used homologous arm is short, and the vector is easily established; and after the exogenous objective albumen is directly integrated in an albumin expression albumen high-efficiency promotor, the expressional specificity of the exogenous albumen of the transgenetic offspring is high, the yield of albumen is high. Moreover, the method also has the advantages of simple operation, safety, no pathogenicity, low immunogenicity and less cell toxins.

The invention discloses a pair of transcriptional activation subsample effect factor nucleases and a coding gene and application thereof. The pair of transcriptional activation subsample effect factor nucleases (TALEN) is obtained by merging a pair of DNA identifying proteins with two different source subunits of a Fok1 DNA incision enzyme respectively, and two adjacent locuses on a prion protein gene (PRNP) exon2 of a goat or a sheep can be identified in specificity mode. When the pair of transcriptional activation subsample effect factor nucleases is simultaneously transferred into a host cell, the nucleases can shoot targets of the exon2 locuses of a host cell PRNP gene and enable the target shooting locuses to have genic mutation so as to perform targeted modification on the PRNP gene of the goat or sheep. The nucleases have the advantages of being strong in specificity, high in target shooting efficiency and accuracy and the like.

The invention provides an automatic target changing device for inertial confinement fusion, belonging to the field of experimental equipment. The invention overcomes the following problems that an existing inertial confinement fusion experiment is long in target changing period and low in target shooting efficiency. The device comprises a translation mechanism, a target library rotating mechanism, a target holder, a target seat, a target changing vacuum cavity and a target pose adjusting device, wherein the translation mechanism is installed inside the target changing vacuum cavity; the target changing vacuum cavity is connected with a vacuum chamber through a vacuum valve; the target seat is installed on the target pose adjusting device; and the translation mechanism comprises a mounting base, a screw rod, a target library sensor, a translation table, a drag chain, a first vacuum motor, a stroke reset sensor and a guide rail, the screw rod and the guide rail are both mounted on the mounting base, the translation table is fixed on a sliding block of the guide rail, the screw rod is connected with the translation table through a nut seat, and the first vacuum motor drives the screw rod to rotate through a gear set. The automatic target changing device is mainly used for automatic target changing of inertial confinement fusion.

The invention relates to ZMIZ1-mediated targeting knockout transcription activator-like effector nuclease, a preparation method and applications, and concretely relates to an artificially synthetic method for a transcription activator-like effector nuclease (TALEN) gene which can target human endogenous ZMIZ1 gene efficiently, and a directed targeting method of recombinant plasmids containing the TALEN gene. The transcription activator-like effector nuclease contains a pair of transcription activator-like effector (TALEs) proteins and catalytic subunits, of DNA endonucleases, which are fused with the proteins respectively, and has functions of identifying and cutting two adjacent loca of the human ZMIZ1 gene respectively. Based on the design of the amino acid sequence of TALEs, the nucleotide sequence coding the TALEN is synthetized and the carrier containing the nucleotide sequence is constructed. Through transfection of cells by utilization of TALENs plasmids, the targeting efficiency of cells can be raised greatly.

The invention provides a method for breeding broad-spectrum bacterial blight-resistant rice by editing the exon of the OsSWEE14 gene. The method is as follows: editing target sequences of first exon and third exon regions of the OsSWEE14 gene to make the target sequences generate mutations of insertion or deletion of a plurality of bases to obtain the broad-spectrum bacterial blight-resistant rice. The target sequence of the first exon region is as shown in the SEQ ID No.1, and the target sequence of the third exon region is as shown in the SEQ ID No.2. The bacterial blight-resistant rice bredby the method of the invention can resist dozens of types of bacterial blight, and has the advantage of wide resistance spectrum.

The invention discloses a nucleotide sequence for repairing DMD gene mutation and a system. A gene editing system can knock out a Dystrophin genomic region between the 31792310th site and the 31854834th site, or between the 31747866th site and the 31838091th site, or between the 31747866th site and the 31854834th site of X chromosome, and preferably, the gene editing system can knock out a Dystrophin genomic region between the 31815201th site and the 31846518th site, between the 31769972th site to the 31815200th site, or the 31769972th site to the 31846518th site of the X chromosome. The geneediting system can target more than 17% of DMD patients, EX51 (deficiency) 4%, EX50 (deficiency) 13%, EX45-50 (deficiency), EX48-50 (deficiency), EX50 (deficiency), EX51 (deficiency), EX52 (deficiency) and EX50 or EX51 exon area point mutation and mutation with various other forms are included, and the adaptability is higher.

The invention relates to the technical field of molecular biology and biomedicine, in particular to a method for preparing a retinopathy non-human mammal model and an application of the mammal model.The method comprises the following steps: knocking out a partial sequence of a second exon of an animal CNGA1 gene by using a Crispr-Cas system, wherein the Crispr-Cas system comprises sgRNA and Cas9enzyme, and the sgRNA comprises sgRNA1 and sgRNA2 of which the corresponding target site sequences are shown as SEQ ID NO: 1 and SEQ ID NO: 2.

The invention belongs to the technical field of engines, and particularly relates to a cooling nozzle and a method for improving the targeting efficiency. The cooling nozzle comprises a spray head anda nozzle, wherein a first channel is arranged in the spray head, a connecting piece is arranged on the spray head, and a second channel communication with the first channel is arranged in the connecting piece; one end of the nozzle is mounted on the connecting piece, the nozzle is in communication with the second channel, and a streaming piece is arranged in the other end of the nozzle. The engine oil in the cooling nozzle flows from the first channel to the second channel and then the engine oil is subjected to flow rate adjusting passing through the streaming piece, and the flow rate of theengine oil in the piston cooling nozzle is subjected to flow rate adjusting by arranging the streaming piece, so that the flow rate in the section of a pipe of the cooling nozzle is more uniform, sothat the targeting efficiency is improved.

The invention discloses a dead SaCas9-Fok1 system, as well as a construction method and application thereof. Due to the fact that the molecular weight of SaCas9 is small, p-dead SaCas9-Fok1 recombinant plasmids use dead SaCas9 to replace traditional dead SpCas9, and the influence of SpCas9 steric hindrance on mutual combination of Fok1 is reduced, so that the targeting efficiency is improved; thedead SaCas9-Fok1 system needs the combined action of Sa-sgRNA in cooperation with 2 21nt, the Sa-sgRNA is longer than Sp-sgRNA(20nt), and the PAM sequence recognized by the SaCas9 is 6nt and longer than PAM(3nt) of SpCas9, so that when the dead SaCas9-Fok1 is matched with two Sa-sg RNAs for use, the off-targeting efficiency is further reduced.

The invention discloses transcriptional activation subsample effect factor nuclease (TALENs) and a coding gene and application thereof. The pair of TALENs are formed by fusing transcriptional activation subsample effect factors (TALEs) of specific sequences on specific recognition mouse PPARgamma2 genes and Fok1 protein, the pair of TALENs are shifted into a mouse cell system at the same time, specificity target shooting can be performed on PPARgamma2 genes of host cells, the target shooting rate is high, and the target shooting efficiency is high.