59 results about "CRISPR/Cpf1" patented technology

The invention discloses applications of a CRISPR / Cpf1 system in gene editing. CrRNA in the CRISPR / Cpf1 system is modified crRNA; the modification manner is desoxyribonucleic acid modification; the desoxyribonucleic acid modification method comprises the step of increasing 1-3 desoxyribonucleic acid at the 5' terminal and / or increasing 1-3 desoxyribonucleic acid at the 3' terminal of crRNA; and the desoxyribonucleic acid is deoxythymidine acid. The experiment proves that the crRNA formed through chemical synthesis and having the modification can cause the mutation of an hAAVS1 gene and a THUMPD3-AS1 gene when used for the AsCRISPR / Cpf1 system or the FnCRISPR / Cpf1 system, namely, the modified crRNA has certain gene editing capacity when used for the AsCRISPR / Cpf1 system or the FnCRISPR / Cpf1 system, and therefore, the modified crRNA has a significant application value in gene editing utilizing the CRISPR / Cpf1 system.

The invention discloses application of a CRISPR (clustered regularly interspaced short palindromic repeats) / Cpf1 system in gene editing. The CRISPR / Cpf1 system comprises a1), chemosynthetic crRNA or a2), a crRNA expressing carrier. The CRISPR / Cpf1 system can be c1), an LbCRISPR / Cpf1 system, c2), an Lb2CRISPR / Cpf1 system, c3), an FnCRISPR / Cpf1 system or c4), an AsCRISPR / Cpf1 system. Experiments prove that the chemosynthetic crRNA can play an effective role in guidance of Cpf1 cut-editing specific target sites, and efficiency is high. Applying the crRNA expressed by the carrier or the chemosynthetic crRNA to the CRISPR / Cpf1 system has high application value in gene editing.

The invention discloses a CRISPR / Cpf1 gene editing system and application of the system in mycobacteria. The CRISPR / Cpf1 gene editing system comprises a recombinant vector carrying optimized FnCpf1 genes and pCR plasmids, wherein the sequence of the optimized FnCpf1 genes is shown in SEQ ID No:1, and the recombinant vector is a colibacillus-mycobacterium shuttle vector containing recombinant enzyme gp60 and gp61 genes; the pCR plasmids contain promoter-driven direct repeat sequence-spacer sequence-direct repeat sequence units, wherein the spacer sequence contains target sequence connection sites. When the CRISPR / Cpf1system is used in the mycobacteria for CRISPR / Cpf1 assisted homologous recombination, high recombination efficiency can be obtained, and gene editing of the mycobacteria can be achieved.

The invention discloses an application of virus-mediated Cpf1 protein in a CRISPR / Cpf1 gene editing system. The CRISPR / Cpf1 gene editing system provided by the invention consists of a recombinant virus or a recombinant vector. The recombinant virus is capable of expressing a Cpf1 gene; and the Cpf1 gene is capable of encoding the Cpf1 protein. The recombinant vector comprises a virus genome and the Cpf1 gene. The virus is either lentivirus or adenovirus. Experiments prove that virus-mediated FnCpf1 protein or AsCpf1 protein causes mutation of an hRb1 gene, an hP53 gene, an hDicer1 gene, an mRb1 gene, an mP53 gene, an mDicer1 gene, an rRb1 gene, an rP53 gene and an rDicer1 gene in the CRISPR / Cpf1 gene editing system. Therefore, the virus-mediated Cpf1 protein is applicable to gene editing in the CRISPR / Cpf1 gene editing system; and the Cpf1 protein has an important application value.

The invention discloses application for using an LbCpf1 - RR mutant in a CRISPR / Cpf1 system in plant gene editing. The application for using the LbCpf1 - RR mutant in the CRISPR / Cpf1 system in plant gene editing uses an OsPDS gene and an OsSBEIIb gene as target genes, constructs a target double loci of one gene and a series of vectors of two genes, and utilizes an agrobacterium-mediated transformation method to import the vectors into rice callus, and rice plants with the target gene knockout are successfully obtained by using the LbCpf1-RR mutant. The only difference between the LbCpf1-RR mutant and the protein LbCpf1 is that the 532nd amino acid changes from G to R, and the 595th amino acid changes from K to R. The LbCpf1-RR mutant provided by the application for using the LbCpf1 - RR mutant in the CRISPR / Cpf1 system in plant gene editing expands the PAM site sequence identified by the LbCpf1-RR mutant, so that the editing range of the CRISPR / Cpf1 system in rice genome is expanded, great significance for promoting the application of the system in the field of plant genome editing is achieved. The application for using the LbCpf1 - RR mutant in the CRISPR / Cpf1 system in plant geneediting has great application value.

The invention discloses a method for regulating expression of an individual CD2AP down. The method comprises the following step of applying CD2AP down-regulation compatibility to the individual, wherein the CD2AP down-regulation compatibility works through siRNA / shRNA, Crispr / cas9, crispr / cpf1, Talen or ZFNs, and therefore, the expression of the CD2AP in a liver tissue of the individual is regulated down.

The invention relates to a Cpf1-based DNA in-vitro splicing method. According to the method, a specific crRNA sequence capable of being recognized by CRISPR-Cpf1 is designed and synthesized and is utilized for guiding Cpf1 to specifically cutting off a specific position of double-stranded DNA and generate preset cohesive ends. By virtue of the method, the preset cohesive ends can be conveniently,rapidly and accurately acquired and are utilized for splicing DNA; and engineering, standardized and modularized remolding or assembling of DNA can be realized.

The invention discloses a method for obtaining label-free transgenic plant. The method is implemented by a CRISPR / Cas9 or CRISPR / Cpf1 system, and a vector of the CRISPR / Cas9 or CRISPR / Cpf1 system comprises a gene edited protein gene, a sgRNA of targeting the vector and a specific temporal and spatial expression gene promoter. The specific temporal and spatial expression gene promoter is not expressed during callus period, and the gene edited protein gene is expressed after genetic transformation is completed. The method provided by the invention has simple operation, and mainly aims at cropsor economic plants, especially plants that are perennial and vegetative propagated; large fragments of transgenic components are lost by splicing the vector, and the significance is great.

The invention discloses a CRISPR / Cpf1 gene editing method applicable to a marine crustacean mitochondrion genome. The method includes the steps: introducing MLS signals positioned by a marine crustacean mitochondrion into a CRISPR / Cpf1 expression plasmid; designing gRNA; micro-injecting fertilized eggs; culturing the fertilized eggs in an in-vitro manner; detecting editing effect to complete editing of the marine crustacean mitochondrion genome. By the method, the marine crustacean mitochondrion genome can be directionally edited, and the method has significant value to a condition that artificial proliferation releasing markers are introduced into the marine crustacean mitochondrion genome.

The invention discloses a CRISPR / Cpf1 system-mediated homologous recombination method using an RNA transcript as a repair template. The method adopts rice ALS genes as a research object, and constructs a homologous recombination vector. RCR1-RCR2-RDR fragments are transcribed in vitro, a RNP method is used, the RNA transcript is used as the repair template, and homologous recombination repair of target genes is achieved in rice callus. The vector is introduced into the rice callus by a gene gun method, and rice plants with ALS gene fixed-point modification are obtained. The results show that RNA used as the repair template can successfully mediate homologous recombination of the target genes, and the method provides a novel idea for crop breeding, and has strong application potential in agricultural breeding.

The invention discloses a bacillus subtilis polygene editing and expression adjusting and controlling system based on CRISPR Cpf1. Through a constructed Cpf1 expression vector pHT-XCR6 and a constructed crRNA array expression vector pcrF11, complete knockout of 2 genes, basic group modification of 6 genes and knock-in of 1 gene can be completed at one time. The vector pHT-XCR6 contains NgAgo protein, and is used for promoting recA mediated homologous recombination. Besides, a vector pLCg6-dCpf1-remA (used for integral expression of a Cpf1 mutant dCpf1 inactivated by deoxyribonuclease and fusion protein of an activating transcription factor remA on a bacillus subtilis genome) and a vector pcra3 (used for integral expression of a crRNA array on the bacillus subtilis genome) are constructed,and can be used for performing transcription inhibition and activation on different genes. The invention further establishes an economic efficient crRNA array assembling method of which the name is SOMACA (Synthetic Oligos Mediated Assembly of crRNA Array), and through synthesizing short-single-strand DNA (60nt ), a needed crRNA array is inserted in the vector pcrF11 or pcra3 and is used for guiding Cpf1 or dCpf1-remA for gene editing and expression adjustment and control.

This invention pertains to recombinant AsCpf1 and LbCpf1 nucleic acids and polypeptides for use in CRISPR / Cpf1 endonuclease systems and mammalian cell lines encoding recombinant AsCpf1 or LbCpf1 polypeptides. The invention includes recombinant ribonucleoprotein complexes and CRSPR / Cpf1 endonuclease systems having a suitable AsCpf1 crRNA is selected from a length-truncated AsCpf1 crRNA, a chemically-modified AsCpf1 crRNA, or an AsCpf1 crRNA comprising both length truncations and chemical modifications. Methods of performing gene editing using these systems and reagents are also provided.

A system and method for reducing the likelihood of colorectal cancer in a human being includes the modification of an individual's gut microbes by employing a Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated system (CRISPR-Cas) or Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (CRISPR / Cpf1) system to modify only bacterial genes of bacteria that reside in the human gut that are non-homologous to those encompassed in the human genome, and in particular, to administer a therapeutically effective amount of a bacterial formulation comprising F. prausnitzii that has been modified to produce one of alliin or butyrate.

A system and method for reducing the likelihood of GERD includes the modification of an individual's gut microbes in a manner that reduces, if not eliminates, the symptoms of GERD. Certain embodiments employ Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated system (CRISPR-Cas) or Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (CRISPR / Cpf1) system to render H. Pylori more susceptible to certain drugs, including antibiotics, thus addressing the antibiotic resistance otherwise experienced by treating H. Pylori with antibiotics.

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.

A method for reducing the likelihood of developing liver cancer in an individual diagnosed with non-alcoholic fatty liver disease that involves providing an individual with an effective amount of a composition of bacteria modified using a Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated system (CRISPR-Cas) or Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (CRISPR / Cpf1) system so that the bacteria is able to produce a therapeutically effective amount of anti-bodies to oxidized low density lipoprotein, with the modified bacteria preferably being from the Lactobacillus, Bifidobacterium, and Streptococcus species; and most preferably including L. reuteri bacteria modified using CRISPR-Cas and / or Cpf1 systems so that it is able to survive the conditions in the duodenum and jejunum of the small intestine of a human.

The invention provides a method of knocking out T cell immunity detecting point pathway genes by using CRISPR-Cpf1 / sgRNA rapidly, simply, efficiently and specifically. The method effectively solves the defects occurring when a CRISPR-Cas9 system knocks out PD-1, CTLA-4, LAG-3 and TIM3 genes. The method comprises the following specific steps: identifying a target DNA region of T cell immunity detection point pathway genes, designing an sgRNA primer pair according to the selected target DNA region, and specifically and directionally knocking out the T cell immunity detection point pathway genesby using the CRISPR-Cpf1 system. The method is simple and practicable, and has relatively good effectiveness on knock-out of T cells through an immunity detection point and relatively high safety.

This invention pertains to recombinant AsCpf1 and LbCpf1 nucleic acids and polypeptides for use in CRISPR / Cpf1 endonuclease systems and mammalian cell lines encoding recombinant AsCpf1 or LbCpf1 polypeptides. The invention includes recombinant ribonucleoprotein complexes and CRSPR / Cpf1 endonuclease systems having a suitable AsCpf1 crRNA is selected from a length-truncated AsCpf1 crRNA, a chemically-modified AsCpf1 crRNA, or an AsCpf1 crRNA comprising both length truncations and chemical modifications. Methods of performing gene editing using these systems and reagents are also provided.

The invention discloses bacillus subtilis with inactivated extracellular protease as well as a construction method and application the bacillus subtilis. B.subtilis 168 is taken as an original strain, a transcription factor comK-comS gene controlled by a xylose inducible promoter PxylA is integrated on a genome by using a CRISPR / Cpf1 technology, so that the transformation efficiency of the bacillus subtilis is greatly improved; a trpC gene and a gudB gene are subjected to reverse mutation, so that tryptophan can be synthesized, and glutamic acid family amino acids can be effectively utilized; and besides, six extracellular protease genes are knocked out, so that the metabolic pressure of cells is reduced, and recombinant proteins can be effectively accumulated outside the cells. The content of extracellular sfGFP produced when a final engineering strain G601 is used for fermentation is 1.88 times of that of the B.subtilis 168, and the total content of intracellular and extracellular sfGFP is 1.94 times of that of the B.subtilis 168. An engineering bacterium constructed by the invention is simple and convenient to transform, has no nutritional deficiency, has better amino acid utilization capacity and low extracellular protease activity, can be widely applied to secretory expression of a recombinant protein, and has a wide application prospect.

The invention discloses a novel method for gene assembly using improved CRISPR-Cpf1. The method includes utilizing a DNA gene editing enzyme FnCpf1 from a Francisella tularemia noticed U112 strain (gene sequence number: MF193599) for gene recombinant cloning. The seed sequence of crRNA (an RNA sequence generated by transcription of regular clustering spaced CRISPR) from 23 nucleotides reported inthe literature to 18 without affecting the FnCpf1 cleavage efficiency. At the same time, oligo(dN)6 (oligonucleotide hexamer) is used as the viscous end of the cleavage target site, the problem of terminal heterogeneity after FnCpf1 digestion is solved, the assembly efficiency of exogenous DNA fragments is remarkably improved, and the DNA fragment assembly efficiency reaches about 85%. The gene editing enzyme FnCpf1 can be replaced with other nucleic acid editing enzymes Cpf1 such as AsCpf1 and LbCpf1.

A system and method for reducing the likelihood of colorectal cancer in a human being includes the modification of an individual's gut microbes by employing a Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated system (CRISPR-Cas) or Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (CRISPR / Cpf1) system to modify only bacterial genes of bacteria that reside in the human gut that are non-homologous to those encompassed in the human genome, and in particular, to administer a therapeutically effective amount of a bacterial formulation comprising F. prausnitzii that has been modified to produce one of alliin or butyrate.

The invention belongs to the field of gene editing, and particularly relates to a FokI and dCpf1 fusion protein expression vector and a fixed-point gene editing method mediated by the vector. The vector fuses mutated and inactivated dCpf1 protein in a CRISPR / Cpf1 editing system with FokI nuclease to construct an efficient gRNA-mediated gene fixed-point editing system. According to the method, thesite-specific action of dCpf1 and the shearing action of nuclease FokI are utilized, and shearing is performed in a dimer form, so that the specificity of the gene editing system can be improved, anda relatively low off-target rate is achieved. Compared with Cas9 protein, the Cpf1 protein has greater advantages for gene editing and other operations. The invention provides the fusion-protein-mediated gene fixed-point editing method which is more convenient and efficient.

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

The present invention relates to optimized systems for the genome editing of eukaryotic cells, preferably plant cells. Also provided is a plant delivery system comprising at least one Cpf1 enzyme, ora plant optimized construct encoding the same, to be synergistically combined with a Cpf1 guide RNA system. Further provided is a Cpf1 guide RNA system being flanked by a Hammerhead ribozyme sequenceat the 5' end and by a plant-derived Hepatitis Delta Virus (HDV)-like ribozyme sequence at the 3' end, or being embedded within a coding or non-coding region, of a sequence encoding a frame sequence.Novel plant-derived HDV-like ribozyme sequences are also provided. Further provided are methods for improved genome editing, and the use of the various systems provided herein to obtain transformed plants, plant cells, tissues, organs, or a progeny thereof, or a plant material, modified in a targeted way even at difficult to access target sites.

The invention belongs to the field of biotechnology and plant molecular biology, particularly relates to a method for separating protoplasts of different tissues and organs of peanuts and an application of the method. In order to facilitate separation of the protoplasts of the different tissues and organs of the peanuts, the invention provides the method for separating the protoplasts of the different tissues and organs of the peanuts according to specific structures of tissue cells of the peanuts. The method is a universal method suitable for separating different peanut tissue cells; protoplast cells of different peanut tissues and organs can be rapidly and efficiently prepared; the separated peanut protoplasts are high in yield and good in cell activity; and the method can be applied topeanut molecular biology related researches such as fusion of peanut somatic cells, development of a peanut protein transient expression system, sequencing of peanut single cells and knockout target detection based on CRISPR / Cas9 and CRISPR / Cpf1.

The present invention relates to a composition for genome editing using a CRISPR / Cpf1 system and a use thereof and, more particularly, to a composition for genome editing comprising: a CRISPR RNA (crRNA) including a guide sequence capable of hybridizing with a target nucleotide sequence, and a uridine repeat sequence connected to the 3′-end of the guide sequence, or a DNA encoding the same; and a Cpf1 protein or a DNA encoding the same, a method for genome editing using the same, a method for construction of a genetically modified organism, and a genetically modified organism. The present invention can increase an indel efficiency and decrease off-target activity in genome editing of eukaryotic cells using the CRIPSPR / Cpf1 system and thus can easily construct a genetically modified cell or genetically modified animal or plant having a desired gene inserted thereinto (knock-in) or deleted therefrom (knock-out).

The invention discloses a CRISPR / Cpf1 vector applicable to deep sea fungi FS140 as well as a construction method and application of the CRISPR / Cpf1 vector. According to the invention, the CRISPR / Cpf1 technology is utilized for the first time to construct a gene knockout system suitable for the deep-sea fungus G.pallida FS140, so that genetic engineering transformation of the G.pallida FS140 is promoted, and a molecular biology foundation is laid for clarification of a biosynthesis mechanism of G.pallida FS140 gliotoxin and acquisition of more gliotoxin derivatives with remarkable biological activity.

Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. The disclosure reports CRISPR / Cpfl -mediated gene editing (Myo-editing) is effective at correcting the dystrophin gene mutation in the mdx mice, a model for DMD. Further, the disclosure reports optimization of germline editing of mdxmice by engineering the permanent skipping of mutant exon and extending exon skipping to also correct the disease by post-natal delivery of adeno- associated virus (AAV). AAV-mediated Myo-editing canefficiently rescue the reading frame of dystrophin in mdx mice in vivo. The disclosure reports means of Myo-editing-mediated exon skipping has been successfully advanced from somatic tissues in miceto human DMD patients- derived iPSCs (induced pluripotent stem cells). Custom Myo-editing was performed on iPSCs from patients with differing mutations and successfully restored dystrophin protein expression for all mutations in iPSCs-derived cardiomyocytes.