[go: up one dir, main page]

WO2019184655A1 - Application de système crispr/cas dans l'édition de gènes - Google Patents

Application de système crispr/cas dans l'édition de gènes Download PDF

Info

Publication number
WO2019184655A1
WO2019184655A1 PCT/CN2019/076784 CN2019076784W WO2019184655A1 WO 2019184655 A1 WO2019184655 A1 WO 2019184655A1 CN 2019076784 W CN2019076784 W CN 2019076784W WO 2019184655 A1 WO2019184655 A1 WO 2019184655A1
Authority
WO
WIPO (PCT)
Prior art keywords
plasmid
trna
sgrna
cell
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2019/076784
Other languages
English (en)
Chinese (zh)
Inventor
汪沛
牛志杰
林彦妮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cure Genetics Co Ltd
Original Assignee
Cure Genetics Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cure Genetics Co Ltd filed Critical Cure Genetics Co Ltd
Priority to CN201980022756.7A priority Critical patent/CN111971389A/zh
Publication of WO2019184655A1 publication Critical patent/WO2019184655A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the invention relates to a DNA expression system and a genetic engineering expression method of a gene editing system, in particular to an expression system of CRISPR/Cas9 and a method for expressing the same.
  • CRISPR/Cas9 CRISPR/Cas9 technology is widely used in fixed-point editing of genomes. Compared to other traditional gene editing tools, such as zinc finger (Bibikova et al. Science 2003. 300: 764), transcription activator-like effector nuclease (TALEN) (Boch et al. Science 2009. 326: 1509–1512), CRISPR/Cas9
  • TALEN transcription activator-like effector nuclease
  • Cas9 is a nuclease that, when combined with single-stranded guide RNA (sgRNA), can specifically target the double-stranded DNA of the genome and cause a double-strand break (DSB).
  • sgRNA single-stranded guide RNA
  • the nuclease Cas9 is capable of specifically targeting DNA in the single-stranded guide RNA (sgRNA) to which it binds.
  • sgRNA single-stranded guide RNA
  • Cas9 and different guide RNAs can be pre-mixed in vitro and then introduced into cells or tissues to achieve multi-site editing (Haoyi et al. Cell 2013. 153:1–9).
  • Cas9 protein and sgRNA are not only expensive, but also greatly limits the method of introduction into the body, which brings great inconvenience to scientific research and industrial production.
  • Cas9 can also be transferred into cells in the form of mRNA or DNA, where the mRNA is very expensive. Therefore, transferring to the Cas9 expression element in the form of DNA is the most cost-effective method.
  • sgRNA can be transferred into cells or tissues in the form of RNA or DNA.
  • Cas9 is in the form of DNA
  • sgRNA is introduced in the form of RNA
  • it is very easy to degrade before Cas9 begins to express so that when Cas9 nuclease is ready, there may be a lack of suitable sgRNA to work with it. Therefore, both are introduced in the form of DNA to more closely match the mode of action of this system.
  • DNA delivery is compatible with viral packaging dip patterns (Malina et al. Methods Enzymol 2014. 546: 193-213), which greatly expands the use of CRISPR/Cas9 for editing in the genome.
  • the efficiency of Cas9 endonuclease multi-localization editing can be achieved.
  • the Csy4 system US 2016/031928 A1
  • introducing a specific RNA recognition and cleavage enzyme, and adding a specific RNA recognition site to the design of the guide RNA although this system can act in the organism, but it will cause Some inconveniences, first of all, the introduction of this system increases the burden of the exogenous DNA of the organism, and the processed guide RNA has an RNA recognition site more than the original structure, which is not required for the downstream reaction.
  • ribozyme sequences that can be self-cleaved at both ends of the gRNA sequence (Yangbin et al. JIPB 2014.56: 343-349), which can release sgRNA by self-cleavage function after transcription in vivo, but this method is limited.
  • the ribozyme can only cleave one end, so that the sgRNA sequence is ligated with different ribozyme sequences at both ends, and it is not convenient to connect DNA fragments corresponding to different guide RNAs; another method is to connect multiple guide RNAs through tRNA, so that After expression in vivo, this long transcript releases a number of different guide RNAs via the in vivo tRNA processing system, which has been validated in both rice and fruit flies (US2016/031928 A1).
  • the invention provides a method for producing a multi-primary single-stranded guide RNA (sgRNA) in a recipient cell, the method comprising obtaining a polynucleotide construct comprising a tandem unit encoding two or more sgRNAs and tRNAs
  • the DNA sequence is introduced into a recipient cell, and the tRNA processing system of the recipient cell cleaves the heterologous polynucleotide at both ends of the tRNA to produce a single-stranded guide RNA (sgRNA).
  • the single-stranded guide RNA (sgRNA) of the present invention is directed against the T cell receptor constant region gene (TRAC), the histocompatibility complex gene (B2M), and the epidermal growth factor receptor gene (EGFR), respectively.
  • sgRNA T cell receptor constant region gene
  • B2M histocompatibility complex gene
  • EGFR epidermal growth factor receptor gene
  • the tRNA of the invention is a sequence which can form a stem-loop structure of a tRNA, including tRNAs which can transport different amino acids, and tRNA sequences derived from different species.
  • the recipient cell of the invention is a mammalian cell, such as 293T, K562, Jurkat, Hela cells, and the like.
  • the invention further relates to a method of producing a multivariate single-stranded guide RNA (sgRNA) and a Cas protein in a recipient cell, comprising a sequence for expression of a versatile sgRNA element, a design guide for a variable region, and a flow method for one-step construction.
  • sgRNA single-stranded guide RNA
  • the invention further encompasses a nucleic acid construct for producing a multi-primary single-stranded guide RNA (sgRNA) comprising: a DNA sequence encoding two or more sgRNAs in tandem with tRNA.
  • sgRNA multi-primary single-stranded guide RNA
  • tRNA sequences are added at the end of the backbone region of each single-stranded guide RNA and then ligated directly.
  • the framework region of the guide RNA is fused to the tRNA sequence as a tandem unit template, wherein the region to which the two are joined contains a leader sequence of the tRNA, typically six bases.
  • primers are designed with a plurality of specific regions of the guide RNA to amplify the template to form a tandem unit.
  • tandem units are sequentially assembled by Gibbson ligation, ligated to the promoter, and more preferably, ligated into a linearized plasmid containing the promoter sequence.
  • a method of expressing a CRISPR-Cas system in a cell comprising:
  • n is an integer between 1 and 8;
  • step (b) transferring the DNA fragment of step (a) into the cell
  • the sgRNA expressed in the cell binds to the Cas protein in the cell to form a CRISPR-Cas system.
  • step (a) is a DNA plasmid.
  • sequence expressing the sgRNA is a 5'-(X)m-constant region backbone sequence-3', wherein X is selected from any one of A, U, C and G
  • sgRNA is capable of specifically binding to a B2M gene, a TRAC gene or an EGFR gene.
  • tRNA is selected from the group consisting of human-chr17.tRNA2-6-GlyGCC, maize-chr9.trna85-GlyGCC, human-chr1.tRNA34-GlyGCC and human-chr17.tRNA41- One of the SerCGAs.
  • a plasmid comprising the DNA fragment expressing (sgRNA-tRNA)n in the first aspect, wherein n is an integer between 1 and 8.
  • n 1 or 2 or 3.
  • tRNA is selected from the group consisting of human-chr17.tRNA2-6-GlyGCC, maize-chr9.tRNA85-GlyGCC, human-chr1.tRNA34-GlyGCC, and human-chr17.
  • tRNA41-SerCGA One of tRNA41-SerCGA.
  • the CRISPR/Cas protein expression system of the invention can quickly realize the assembly of multiple sgRNA elements in a plasmid, and can simultaneously target multiple targets for efficient editing in vivo, and the invention can also provide a universal platform for testing different connections. The efficiency of processing the sgRNA in the component body.
  • Figure 1 Schematic diagram of the STS-pUC57 plasmid
  • sgRNA1 targets gene TRAC
  • sgRNA2 targets gene B2M
  • tRNA is human-chr17.tRNA2-6-GlyGCC (hereinafter replaced by tRNA gly )
  • STS, STST, TSTS and TSTST indicate that there are different elements between the promoter of the plasmid U6 and the terminator;
  • the STS element is sgRNA1-tRNA gly- sgRNA2;
  • the STST element is sgRNA1-tRNA gly- sgRNA2-tRNA gly ;
  • TSTS element For tRNA gly -sgRNA1-tRNA gly -sgRNA2;
  • TSTST element is tRNA gly -sgRNA1-tRNA gly -sgRNA2-tRNA gly ;
  • S2TS1T element is sgRNA2-tRNA gly -sgRNA1-tRNA gly ;
  • +Cas9 is co-transformed plasmid expressing SpCas9 ;STST-Cas9 is a single plasmid that can simultaneously express STST elements as well as Cas9 proteins.
  • S1-Cas9 a plasmid capable of expressing sgRNA1 and Cas9, editing the target TRAC gene;
  • S2-Cas9 a plasmid capable of expressing sgRNA2 and Cas9, editing the target B2M gene;
  • S3-Cas9 a plasmid capable of expressing sgRNA3 and Cas9, editing the target EGFR gene;
  • +Cas9 is a plasmid that expresses SpCas9
  • STST-Cas9 is a single plasmid that can simultaneously express STST elements and Cas9 protein
  • STSTST-Cas9 can simultaneously express STSTST (sgRNA1-tRNA gly- sgRNA2--tRNA gly- sgRNA3- The tRNA gly ) element and the single plasmid of the Cas9 protein.
  • Figure 4 Schematic diagram of the synthesis of self-cutting elements
  • a pair of primers (general forward primer and variable reverse primer) in single-strand synthesis and N-pair primers in element synthesis need to be synthesized by sequence, and other products can be obtained by PCR amplification.
  • Figure 5 Schematic diagram of the starting plasmid and the final construct of the plasmid
  • the linearized plasmid primer design is shown (A) and the linearized plasmid product is shown (B); the linearized plasmid is ligated with the multi-fragment synthesized in Figure 4 to finally form a plasmid construct (C); wherein the plasmid skeleton (Plasmid scaffold) It can be varied, and may contain Cas9 expression elements or elements for lentiviral packaging, in addition to plasmid basic elements including plasmid replication, resistance genes, and the like.
  • FIG. 6 Schematic diagram of secondary structure of tRNA; panels A, B, C and D indicate T (human-chr17.tRNA2-6-GlyGCC); T1 (corn-chr9.tRNA85-GlyGCC); T2 (human-chr1.tRNA34, respectively) -GlyGCC); T3 (human-chr17.tRNA41-SerCGA)
  • Figure 7 Comparison of the editing efficiency of the target multiplexed sgRNA expression elements in 293T cells using the different tRNAs (T, T1, T2 or T3) in Figure 6 as the ligation elements.
  • Figure 8 Comparison of the editing efficiency of the target multi-sgRNA expression elements in K562 cells with different tRNAs (T1, T2 or T3) as the ligation elements in Figure 6.
  • the CRISPR/Cas system is an immune system found in prokaryotes, mainly for genetic components that are invasive, such as plasmids or viruses.
  • the system can be immunized from foreign genetic elements by retaining its DNA fragments in a certain format.
  • the retained DNA can be transcribed into a guide RNA and combined with a single protein or protein complex of the system to directionally recognize and cleave the re-invading genetic elements.
  • the CRISPR/Cas9 system is one of the widely studied branches. Since the effect part of the system involves only a single protein (Cas9 protein), it is more convenient and widely used in scientific research.
  • the clustered regular interval palindrome repeat (CRISPR) and the CRISPR binding protein (Cas protein) constitute a powerful nuclease system.
  • the CRISPR-associated endonuclease Cas protein can target specific genomic sequences via single-stranded guide RNA (sgRNA).
  • Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Cas12, Cas13, Cas14, Csy1, Csy2, Csy3 , Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3 , Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologous proteins in different species, mutant proteins inactivated by endonucleases, or modified forms thereof.
  • the Cas protein of the invention is a Cas9 protein.
  • Cas9 also known as Csn1 or Csx12, is a giant protein involved in both crRNA biosynthesis and in the destruction of invading DNA.
  • S. thermophilus Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and Streptococcus pyogenes Cas9 is described in (S. Pyogenes) (Deltcheva, Chylinski et al. 2011).
  • the Cas9 of the present invention includes, but is not limited to, the following types: Streptococcus pyogenes Cas9 protein, the amino acid sequence of which is described in SwissProt database accession number Q99ZW2; Neisseria meningitidis Cas9 protein, the amino acid sequence of which is described in the UniProt database. No. A1IQ68; Streptococcus thermophilus Cas9 protein, the amino acid sequence of which is shown in UniProt database number Q03LF7; Staphylococcus aureus Cas9 protein, the amino acid sequence of which is shown in UniProt database number J7RUA5.
  • sgRNA Single-stranded guide RNA
  • the guide RNA In the CRISPR/Cas9 system, the guide RNA consists of two parts, one is the crRNA and the other is the trans-crRNA (tracrRNA), which forms a complex by the complementation of the base pair. In practical use, the two can be fused together in the form of a single-stranded guide RNA, which is still capable of forming a structure similar to the previous double strand, and functions to guide the recognition and cleavage of foreign genes.
  • the sgRNA is a single-stranded guide RNA and generally comprises a leader sequence, a tracr pairing sequence and a tracr sequence.
  • the structure of the sgRNA includes the initial -20 base variable region (ie, the leader sequence) and the successive constant region backbone of approximately 80 bases (including the tracr pairing sequence and the tracr sequence, which appear as fused single-stranded forms in the sgRNA). ).
  • the sgRNA is an sgRNA capable of specifically binding to a B2M gene, a TRAC gene or an EGFR gene.
  • Transport RNA (tRNA)
  • a transport RNA is a polyribonucleic acid (RNA) molecule that can transport amino acids, and its mature form is usually in the form of a single strand consisting of 70-90 nucleotides. These single strands can be further formed into a secondary structure in the form of clover, usually by base pairing within the molecule.
  • Transport RNA is present in a variety of living organisms, including bacteria, plants, animals, and the like. It is a tool for connecting messenger RNA and proteins in life. One end of the transfer RNA binds to the messenger RNA through base pairing, translates the genetic information it carries, and the other end links specific amino acids and transports them to the protein synthesis machine in the order of messenger RNA.
  • the genetic information of the organism will be reflected in the amino acid sequence of the protein, and then the various proteins will be the main force for performing life activities. Therefore, the transport RNA is widely existed and maintains a stable and basically consistent structure in evolution.
  • the tRNA is further processed from a transcript transcribed from the genomic tRNA gene (from the 5' end to the 3' end). These processing include removal of a portion of the intron, cleavage at the 5' and 3' ends of the tRNA, tailing at the 3' end, and modification of the base and the like.
  • cleavage at both ends of the tRNA is a property utilized in this patent: in eukaryotes (including plants and animals), RNaseP is mainly used to remove the 5'-end redundant sequence, and RNaseZ is used to remove the 3'-end redundant sequence.
  • the tRNA gene of the invention is derived from human (Homo sapiens) or maize species (Zea_mays).
  • the tRNA gene of the invention is human-chr17.tRNA2-6-GlyGCC (tRNA sequence chr17:8029064-8029134); human-chr1.tRNA34-GlyGCC (chr1:161413094-161413164); human- chr17.tRNA41-SerCGA (chr17:8042199-8042280); maize-chr9.tRNA85-GlyGCC (chr9: 152440635-152440705); preferably human-chr1.tRNA34-GlyGCC.
  • the above gene information can be found from the tRNA website gtrnadb.ucsc.edu/.
  • the CRISPR/Cas system used in the present invention is in the form of DNA and involves the construction of a plasmid.
  • a plasmid is a genetic material that is extrachromosomally present in the cytoplasm. It is usually a closed circular double-stranded DNA molecule that functions as a self-replicating agent and expresses the genetic material it carries.
  • the invention mainly uses a plasmid to introduce into a cell, and plays the following two functions in the cell: the Cas expression system carried by the plasmid (including a promoter, a Cas humanized sequence, a nuclear signal, etc.) can be transcribed and translated into a Cas protein in the cell body.
  • the plasmid-carrying guide RNA expression cassette (cassette) can be transcribed and processed in the cell body to obtain mature single-stranded guide RNA.
  • the DNA sequence which can express (sgRNA-tRNA)n can be constructed in the same plasmid as the DNA sequence of the Cas protein, or can be constructed separately in two plasmids.
  • This plasmid may also be a shuttle plasmid for virus preparation.
  • sgRNA single-stranded guide RNA
  • the tRNA can be processed and released in vivo using the processing system of tRNA in vivo.
  • tRNA-linked guide RNA we have summarized the following patterns.
  • the researchers tend to add tRNA to both ends to improve editing efficiency.
  • the researchers used tRNA-sgRNA as the basic unit to perform multiple concatenations to achieve multi-site targeted editing.
  • the present invention compares the manner and sequence of various sgRNA and tRNA linkages, including direct sequence synthesis, molecular cloning by restriction enzyme ligation, Gibbson assembly, and the Golden Gate method to achieve different constructs.
  • the tRNA that is subsequently ligated can be considered as the backbone extension of the original sgRNA, which is also the constant region.
  • the constant region is used as a template in the present invention, and the sequence of the variable region can be introduced via a PCR reaction through different primers.
  • the template can be directly synthesized into a double strand ( ⁇ 160 nt), or a partially complementary single strand can be synthesized by PCR to form a double strand. Since the sgRNA protoskeleton in the constant region has been optimized, it is not necessary to change this part for the same Cas9 (this skeletal sequence is generally different for different sources of Cas9 protein), and there are many spaces for tRNA selection, including for different species. Different types of tRNA can also select other non-tRNA RNA elements that can be cleaved at both ends.
  • two single strands are synthesized, one comprising only the sgRNA backbone portion and the other comprising different tRNA sequences and portions that overlap a small amount with the first strip.
  • the two single strands are annealed and PCR extended to obtain a double-stranded template; then PCR amplification is performed according to multiple target design primers to obtain multiple sgRNA-tRNA elements.
  • the plasmid vector was linearized and ligated seamlessly with the sgRNA-tRNA element to obtain the plasmid of the desired construction.
  • a DNA plasmid of (sgRNA-tRNA)n is constructed, together with a plasmid which can express the Cas protein, or a single plasmid which can simultaneously express (sgRNA-tRNA) n and Cas protein.
  • the present invention has the following three ways to construct a CRISPR/Cas system in a cell. Transfection methods can be selected from liposome transfection, electroporation transfection (electroporation) and viral infection.
  • Method 1 Liposomal transfection of 293T cells: After 293T cells were resuscitated, cultured to the third generation or above, cells were passaged one day before transfection, and cells were plated in 24-well plates at 50,000 cells per well. When the cells were grown at about 40%-60% of the bottom area of the plate, Lipo2000 (Life 11668-027) was used as a transfection reagent, and a total of 500 ng of plasmid was transfected per well. Editing efficiency was determined 48 hours after cell transfection.
  • Method 2 K562 cells were electroporated, and after cell resuscitation, cultured to the third generation or above, cell counts were performed on the day of transfection, and the mixed cells (200,000) and plasmid (500 ng) were subjected to the following conditions: voltage 1450 V, 10 msec width, 3 pulses. The cells were electroporated for 48 hours and assay efficiency was determined.
  • Method 3 DNA fragments capable of expressing (gRNA-tRNA) n and Cas proteins were cloned into a viral vector, and 293T cells were transfected: one day before transfection, 293T cells were digested into 100 mm culture dishes at a density of 70- 80%, cultured for 24 hours (complete medium: DMEM (Gibco C11995500BT), 10% serum (Corning 35010155), the medium was replaced with Opti-MEM; 32 ⁇ g of the viral vector plasmid to be transfected and the transfection reagent were separately dissolved In 500 ⁇ l Opti-MEM (Gibco 31985-070), the mixture was allowed to stand for 5 minutes, and the transfection reagent was added dropwise to the plasmid, and the mixture was allowed to stand at room temperature for 20 minutes, and the mixture was added to the cell culture medium for 6-8 hours.
  • the fresh complete medium was changed.
  • the culture supernatant was collected for 48 hours and 72 hours, and the supernatant was collected by centrifugation at 3500 rpm for 10 minutes, and the supernatant was filtered through a 0.22 ⁇ m filter.
  • the virus supernatant was subjected to a medium replacement method. Infect other primary cells or cell lines, change the fresh complete medium 6-8 hours after infection, and analyze the target gene editing efficiency of the infected cells after 40 hours of incubation.
  • the TIDE assay is performed in the present invention: the cells are subjected to lipofection, electroporation or virus infection for 48-72 hours, and the cells are repeatedly blown to form a cell suspension, which is collected in an EP tube. The cell suspension was centrifuged and the supernatant was discarded. 80 ⁇ l of DNA extraction reagent QE (Lucigen: QE09050) was added and the cells were mixed. Heat at 98 ° C for 2 minutes and then at 65 ° C for 6 minutes. PCR amplification of the target gene fragment was carried out using this cell lysate as a template. The amplified products were sequenced and the editing efficiency was determined by analyzing the peaks of the editing sites.
  • Beta-2 microglobulin is the light chain of class I MHC molecules and is therefore an integral part of the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • B2M is encoded by the b2m gene located on chromosome 15, while other MHC genes are present as gene clusters on chromosome 6.
  • the human B2M protein has 119 amino acids (see UniProt database code P61769).
  • B2M is essential for the presentation of class I MHC molecules on the cell surface and the stability of the polypeptide binding groove.
  • Class I MHC molecules are present on all nucleated cell surfaces in the human body. Mismatches in MHC can cause immune rejection and cause graft destruction. Elimination of mismatches can be achieved by knocking out B2M genes to remove Class I MHC molecules on the cell surface. .
  • T cell receptor is a receptor on the surface of T cells involved in the activation of T cells after contact of T cells with the presented antigen.
  • TCR consists of two chains, alpha and beta, forming a heterodimer and forming a T cell receptor complex on the cell surface along with the CD3 molecule.
  • Each of the alpha or beta strands contains a variable region and a constant region, wherein the constant region of the alpha chain is encoded by a TRAC gene located on chromosome 14.
  • TCR recognizes a processed polypeptide fragment that binds to an MHC molecule and is also referred to as MHC restriction because recognition requires the presentation of MHC molecules.
  • the TCR When the MHC molecules of the donor and recipient are different, the TCR is able to recognize the difference in MHC and cause activation and expansion of T cells, possibly causing graft versus host disease (GvHD). Knocking out the TRAC gene removes the expression of the TCR alpha chain, thereby removing the TCR from the surface of the T cell, thereby preventing graft-versus-host disease caused by TCR recognition of allogeneic antigen.
  • GvHD graft versus host disease
  • EGFR epidermal growth factor receptor
  • HER1 epidermal growth factor receptor
  • HER2 erbB2, NEU
  • HER3 erbB3
  • HER4 erbB4
  • the HER family plays an important regulatory role in the process of cell physiology.
  • EGFR is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, etc.
  • EGFR signaling pathway plays an important role in the physiological processes such as cell growth, proliferation and differentiation.
  • Example 1 Comparison of editing efficiency of different expression vectors of tRNA-mediated sgRNA in cells
  • EXPERIMENTAL OBJECTIVE To verify the validity of simultaneous expression of multiple sgRNAs, the effect of the number and location of tRNAs on the entire expression element, and the sequence of sgRNA in the expression elements, the validity of the single plasmid system.
  • the test system uses the mammalian cell line 293T, and the editing efficiency of each sgRNA corresponding site is used to compare the effectiveness of different constructs. At the same time, combined with the ease of operation, a highly efficient general model is obtained.
  • sgRNA The components of sgRNA are obtained by gene synthesis
  • the gene was synthesized as follows, wherein the sequence includes a U6 promoter (SEQ ID No: 1), a DNA fragment expressing sgRNA1 (SEQ ID No: 2), a DNA fragment expressing tRNA gly (SEQ ID No: 3), and a DNA fragment expressing sgRNA2. (SEQ ID No: 4) and U6 terminator (DNA fragment: tttttt).
  • sgRNA1-tRNA gly- sgRNA2 is an STS element, wherein S represents sgRNA and T represents tRNA.
  • the gene fragment was subcloned into the pUC57 vector, and the resulting plasmid STS-pUC57 (sequence map is shown in Figure 1) was transformed into competent E. coli (DH5a) and delivered as a bacterial form by the synthesis company (GeneWiz).
  • F represents the forward primer and R represents the reverse primer.
  • PCR was carried out using each of the primers synthesized above using the plasmid STS-pUC57 as a template, and each PCR fragment was subjected to gel purification (QIGEN kit).
  • PCR reaction setup template 10 ng; forward primer (F): 2.5 ⁇ l; reverse primer (R): 2.5 ⁇ l; 2X Q5 (PCR premix, NEB, MO494S): 25 ⁇ l; H 2 O: 40 ⁇ l.
  • the PCR product was separated by 1% agarose gel electrophoresis, and the obtained product was recovered by a kit (QIAGEN 28706), and the final product was dissolved in water and subjected to concentration measurement.
  • TRNA is simultaneously added to both ends of the STS expression element to obtain an expression element of TSTST.
  • the specific construction method is as follows: using the constructed STST-pUC57 as a template, P1V-F and P1V-R are primers for PCR to linearize the plasmid. The reaction settings and program settings are the same as 2.1. The PCR product was purified by gel electrophoresis, and the concentration was measured, and the next assembly was carried out. Gibson assembly: PCR product in 2.2: 100 ng; P1T: 30 ng; 2X Gibson master mix (NEB 2611S): 10 ⁇ l; H 2 O: 20 ⁇ l. Thereafter, the reaction conditions, transformation conditions, sequencing identification and purification were the same as 2.1, and the plasmid TSTST-pUC57 was constructed in this way.
  • S2TS1T refers to the construction of the sgRNA2-tRNA gly- sgRNA1-tRNA gly form: using the constructed STST-pUC57 as a template, the following primers were used for plasmid linearization and fragment element amplification.
  • the digestion reaction of BbsI was set for the PCR product: template (PV, S1T, S2T): 1 ⁇ g; BbsI (NEB R0539V): 1 ⁇ l; NEB Buffer 2.1 (NEB B7202V): 3 ⁇ l; H 2 O: flattened to 30 ⁇ l.
  • the digestion was carried out in a 37 ° C water bath for 2 hours.
  • the digested product was separated by 1% agarose gel electrophoresis, and the obtained product was recovered by a kit (QIAGEN 28706) and the final product was dissolved in water and quantified.
  • the ligation reaction was set: the digested fragment obtained in the previous step (PV: 1 ⁇ g; S1T: 500 ng; S2T: 500 ng) T4 ligase: 0.6 ⁇ l; T4 ligase buffer 1 ⁇ l; H 2 O was filled in 10 ⁇ l.
  • the ligation was carried out at 16 ° C for 2 hours.
  • the ligation product transformed competent cell TOP10 (Tiangen CB104). Pick up the monoclonal culture and sequence it. Monoclonal expansion culture and plasmid extraction as in 2.1.
  • the SpCas9-pX330 plasmid (Addgene 71707) carries the elements required for expression of Cas9 in eukaryotic cells, and as a plasmid backbone, subcloning of the guide RNA expression element into this plasmid enables multi-site editing by a single plasmid system.
  • the plasmid pX330 was linearized by PCR according to the following primers, and the expression elements (collectively referred to as (ST) n) were amplified:
  • the digestion reaction was set: template (pX330L, (ST)n) 1 ⁇ g; AflIII (NEB: R0541V) 1 ⁇ l; XbaI (NEB R0145V) 1 ⁇ l; NEB Cutsmart buffer 3 ⁇ l; H 2 O to fill 30 ⁇ l.
  • the digestion was carried out in a 37 ° C water bath for 2 hours.
  • the digested product was separated by 1% agarose gel electrophoresis, and the obtained product was recovered by a kit (QIAGEN 28706) and the final product was dissolved in water and quantified.
  • the ligation reaction was set: the cleavage fragment obtained in the previous step (pX330L 500 ng; (ST) n 400 ng) T4 ligase: 0.6 ⁇ l; T4 ligase buffer 1 ⁇ l; H 2 O to fill 10 ⁇ l.
  • the ligation reaction was carried out at 16 ° C for 2 hours.
  • the ligation product transformed competent cell TOP10 (Tiangen CB104). Pick up the monoclonal culture and sequence it. Monoclonal expansion culture and plasmid extraction as in 2.1, the final plasmid product is STST-pX330, which can simultaneously express STST elements and Cas9 protein.
  • the above four pairs of primers were used for PCR reaction, BbsI digestion and ligation transformation, and the product STSTST-pUC57 was cloned and identified. See Tables 2.1 and 2.3 for specific experimental conditions. According to the single plasmid construction method in 2.4, the final plasmid product was designated as STSTST-pX330. This construct can express the Cas9 protein and three different sgRNAs.
  • Primer annealing The primers were diluted to 100 ⁇ M and mixed under the following conditions: primer F 1 ⁇ l; primer R 1 ⁇ l; 10*T4 ligase buffer 1 ⁇ l; T4PNK (NEB, M0201S) 0.5 ⁇ l; water supplemented to 10 ⁇ l.
  • the experimental mixture was allowed to react at 37 ° C for 30 minutes, and at 95 ° C for 3 minutes, the resulting product was recovered by a kit (QIAGEN 28706) and the final product was dissolved in water and quantified.
  • Digestion template reaction template SpCas9-pX330 plasmid (Addgene #71707) 3 ⁇ g; BbsI (NEB #R0539S) 3 ⁇ l; NEB Cutsmart buffer 3 ⁇ l; H 2 O to fill 30 ⁇ l.
  • the digestion was carried out in a 37 ° C water bath for 2 hours.
  • the digested product was separated by 1% agarose gel electrophoresis, and the obtained product was recovered by a kit (QIAGEN 28706) and the final product was dissolved in water and quantified.
  • the ligation reaction was set: 100 ng of template after enzymatic cleavage; 2 ⁇ l of primer after annealing; T4 ligase (NEB #M0202L): 0.6 ⁇ l; 1 ⁇ l of T4 ligase buffer; 10 ⁇ l of H 2 O, and ligated at room temperature for 2 hours.
  • the ligation product was subjected to bacterial transformation, and monoclonal sequencing was identified as above.
  • the final plasmid products designated S1-pX330, S2-pX330 and S3-pX330, respectively, can express the Cas9 protein and the corresponding sgRNA.
  • Collecting cells 48 hours after cell transfection, the cells were repeatedly beaten to form a cell suspension, which was collected in an EP tube.
  • Genomic extraction Centrifuge the cell suspension and discard the supernatant. 80 ⁇ l of QE (Lucigen: QE09050) was added and the cells were mixed. Heat at 98 ° C for 2 minutes and then at 65 ° C for 6 minutes.
  • the primer information is as follows
  • PCR settings template: 1 ⁇ l genome; forward primer: 1 ⁇ l; reverse primer: 1 ⁇ l; 2X Amplitag (PCR premix, Thermo, 4398790): 10 ⁇ l; H 2 O: 7 ⁇ l.
  • tRNA-ligated gRNA can efficiently release and mediate downstream gene editing.
  • the tRNA at both ends has less influence than the basic element of STS.
  • the STST build is the object of our choice.
  • Example 2 One-step construction of a self-cleavage guide RNA expression vector
  • Variable tRNA single chain (sgRNA SC B20-XXXXX-tRNA) RC
  • SC indicates the sgRNA backbone and SC B20 indicates 20 bases at the 3' end of the sgRNA backbone.
  • XXXXXX indicates that 6 bases of the tRNA 5' end are closely linked in the genomic sequence.
  • the upstream and downstream sequences of different tRNAs can be searched by http://gtrnadb.ucsc.edu/ on the website; RC indicates the reverse of the sequence in the parentheses. To the complementary sequence.
  • Reaction conditions The above two synthetic single chains were each dissolved in water to a final concentration of 100 ⁇ M.
  • Set the PCR system (general single strand: 2.5 ⁇ l; variable tRNA single strand: 2.5 ⁇ l; 2X Q5 (PCR premix, NEB, MO494S): 25 ⁇ l; H 2 O: 20 ⁇ l); set PCR reaction conditions: 98 ° C, 30 seconds; (95 ° C, 10 seconds; 55 ° C, 20 seconds; 72 ° C, 20 seconds) 35 cycles; 72 ° C: 2 minutes. After the reaction was over, the sample was placed at 4 °C. After purification of the sample by PCR purification kit (QIAquick 154045041), the sample can be stored at -20 ° C as a template for use in a one-step reaction.
  • U6TR 20 indicates 20 bases after the element insertion site on the plasmid, generally including the U6 terminator (tttttt) and the subsequent 14 bases, which are determined according to the specific plasmid.
  • (U6PRO B20) RC indicates that the reverse complement of the last 20 bases of the U6 promoter, S1, S2, S3, ... indicates any single-stranded guide RNA arranged in sequence, and F19 indicates the first 19 bases at the 5' end.
  • Base, RC represents a sequence that is reverse complementary.
  • SC denotes the sgRNA backbone
  • SCN20 denotes 20 bases at the 5' end of the sgRNA backbone
  • B20 denotes 20 bases at the 3' end
  • (tRNA B20) RC denotes a reverse complement of 20 bases at the 3' end of the tRNA.
  • Each pair of forward and reverse primers was synthesized by Genewiz and dissolved in water to a final concentration of 10 ⁇ M.
  • the PCR system is set up as above.
  • the reaction conditions were modified as follows: 98 ° C, 30 seconds; (95 ° C, 10 seconds; 55 ° C, 20 seconds; 72 ° C, carrier length (nt) / 1000 * 30 seconds) 35 cycles (see Figure 4); 72 ° C :5 minutes.
  • the sample was stored at 4 °C.
  • the sample was subjected to 1% agarose gel electrophoresis, and the target band was excised for recovery and purification. After purification, it can be stored at -20 °C.
  • the concentration of the linearized plasmid and the multiple fragments were recovered after gel recovery.
  • the mixture was mixed in the following manner: carrier (300 ng/ ⁇ l), target fragment 1 ⁇ g/strand, 2x Gibson master mix (NEB 2611S): 10 ⁇ l, hydrating to 20 ⁇ l.
  • the reaction mixture was placed at 50 ° C for 2 hours.
  • Transform into competent cell TOP10 (Tiangen CB104). Pick up the monoclonal culture and sequence it.
  • Primer sequencing was designed at positions of about 200 bases upstream and downstream of the fragment assembly, and the cloned clones were expanded and cultured, and the plasmid was extracted by an endotoxin-free plasmid extraction kit (QIAGEN 12362).
  • a vector having a Cas9 expression element such as the SpCas9-pX330 plasmid (Addgene 71707), is used. Linearization is performed in areas that do not affect the function of other elements, and linearized primer design refers to the primer design for the carrier in Method 2.
  • Each pair of forward and reverse primers was synthesized by Genewiz and dissolved in water to a final concentration of 10 ⁇ M.
  • the PCR system was set up: (forward primer: 2.5 ⁇ l; reverse primer: 2.5 ⁇ l; template (plasmid containing Cas9 expression element, such as SpCas9-pX330 plasmid): 10 ⁇ g; 2X Q5 (NEB M0494S)): 25 ⁇ l; H 2 O : 20 ⁇ l); setting PCR reaction conditions: 98 ° C, 30 seconds; (95 ° C, 10 s; 55 ° C, 20 s; 72 ° C, (carrier length / 1000 * 30) s) 35 cycles; 72 ° C: 2 minutes. After the reaction was over, the sample was stored at 4 °C. The sample was subjected to 1% agarose gel electrophoresis, and the target band was excised for recovery and purification. After purification, it can be stored at -20 °C.
  • the linearized vector and the synthetic element in the method 2, using the assembly method in the method 3, obtain a single plasmid which can simultaneously express the Cas9 protein and a plurality of gRNAs.
  • the sequencing primers need to be modified according to the plasmid used, and primers are generally designed at positions of about 200 bases upstream and downstream of the fragment assembly.
  • this multi-guided RNA expression vector is mainly summarized, including the synthesis of template strands, the principle of synthesis of specific primers, and other materials and complete processes required for construction (Fig. 4 and Fig. 5). ).
  • sgRNA-tRNA was used as a self-cleaving element to release mature guide RNA in vivo and to mediate efficient editing of multiple targets.
  • For the assembly of multiple units involves seamless cloning, because there can be no extraneous sequences between the units to affect the cleavage in vivo, which in turn affects the correct processing release of sgRNA. Comparing the methods of different seamless clones, we tend to choose Gibson assembly. Because this method does not require a specific recognition site, and the length of the overlapping sequence it needs is exactly compatible with the length of the variable region, it provides a lot of convenience for subsequent experiments. Thus all of the unit products and the linearized vector can be constructed in one step to obtain the desired plasmid construction. If the original plasmid contains a Cas9 expression element, the final constructed plasmid contains all of the elements of the multi-site targeted editing, and the introduction of a single plasmid can achieve a multi-site editing effect in vivo.
  • the guide RNA has several advantages over the previously constructed construction method: for the conservative structure of the guide RNA, it is equivalent to extending its skeleton sequence, so that it can be processed in the cell and release the mature guide RNA; The method is more direct, from template primer synthesis to completion of the final construction can be completed in a short time, mainly involving PCR reactions and seamless ligation reactions; its construction does not involve enzyme cleavage sites, so for almost all Both sgRNAs are suitable and can be applied to sgRNAs expressing various combinations. And fully combined with the advantages of Gibson assembly, the specific sequence (guide sequence) in the sgRNA becomes the bridge region, thus ensuring that the components can be assembled efficiently and orderly according to our needs.
  • the constant region is a relative definition.
  • the skeleton sequence of the sgRNA used can be changed according to the specific use of the protein, and other designs and operations are unchanged; at the same time, for the components connecting different sgRNAs, It can be used as a screening platform to change the construction and effect evaluation of different connected components by changing one of the template chains.
  • Example 3 is the ability to test the ability of different tRNAs to release guide RNA using this method platform.
  • the tRNA selects tRNA1 (SEQ ID NO: 44), tRNA2 (SEQ ID NO: 45) or tRNA3 (SEQ ID NO: 46), and a schematic representation of the secondary structure of all tRNAs used is shown in Figure 6.
  • the template design requires a 6-base leader sequence (query via the tRNA database http://gtrnadb.ucsc.edu/) to facilitate later tRNA processing.
  • Reverse primers were synthesized for different tRNA templates: tRNA1 reverse primer: (SEQ ID NO: 47); tRNA2 reverse primer: (SEQ ID NO: 48); tRNA3 reverse primer: (SEQ ID NO: 49).
  • the synthesized product is a tRNA1 template, a tRNA2 template, and a tRNA3 template.
  • element synthesis and vector linearization were carried out by means of PCR amplification: STS-pUC57 was selected as the template for vector linearization, and different tRNA templates generated in 1 were selected for the template synthesized by the element, and the primers used were used. As shown in Table 9, the reaction conditions are referred to in Example 2.
  • T1 T2, T3 a plasmid capable of expressing (ST)n, which is ligated to different sgRNAs by different tRNAs (tRNA1, tRNA2, tRNA3), was designated as T1, T2, T3.
  • Example 1 See Method Materials in Example 1 for plasmid transfection and editing efficiency analysis of 293T cells.
  • the plasmid was introduced by electroporation (Thermo MPK5000), the number of cells per reaction system was 10 5 , the cell culture medium was 89% RPMI medium 1640 (Gibco C11875500BT), 10% FBS (Corning 35010155), 1% Pen Step ( Gibco 15140-122).
  • the electrical parameters are as follows: 1450 volts, 10 milliseconds, 3 pulses. After electroporation, the cells were cultured for two days, and the cells were harvested for analysis of gene editing efficiency. The method is identical to the 293T cells, see the method materials in Example 1.
  • Example 2 provides a method for constructing multiple guide RNA co-expression elements that can be applied to rapid screening of elements that link the guide RNA.
  • This connected component mainly separates different sgRNAs and releases the various sgRNAs required in the body, so that the connecting elements need to meet the following requirements: short length, no excessive redundant DNA fragments are introduced; Does not adversely affect the structure of sgRNA; both ends can be precisely cut, relying on endonuclease activity to allow this self-cleaving element to release both ends without causing excessive interference to the sgRNA sequence; Without relying on the introduction of an exogenous endonuclease system, the endonuclease that is present in the cell requires accurate recognition and efficient cleavage activity on the element, or the element has an efficient self-cleavage activity independent of other tools.
  • the tRNA is a connecting element that satisfies the above conditions.
  • This build platform can test the effects of different tRNAs as connecting elements. And according to the platform design and the general process, to test the connection components can use several fixed sgRNA to detect the editing efficiency, only one template single chain needs to be changed.
  • T2 human-chr1.tRNA34-GlyGCC
  • K562 human-chr1.tRNA34-GlyGCC

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé d'expression d'ingénierie génétique pour CRISPR/Cas, comprenant la construction rapide d'un élément pour la co-expression d'une pluralité d'ARN guides au moyen de la construction d'un plasmide comprenant une séquence d'ADN exprimant une pluralité d'ARNsg-ARNt.
PCT/CN2019/076784 2018-03-27 2019-03-02 Application de système crispr/cas dans l'édition de gènes Ceased WO2019184655A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201980022756.7A CN111971389A (zh) 2018-03-27 2019-03-02 CRISPR/Cas系统在基因编辑中的应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810258819 2018-03-27
CN201810258819.6 2018-03-27

Publications (1)

Publication Number Publication Date
WO2019184655A1 true WO2019184655A1 (fr) 2019-10-03

Family

ID=68060942

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/076784 Ceased WO2019184655A1 (fr) 2018-03-27 2019-03-02 Application de système crispr/cas dans l'édition de gènes

Country Status (2)

Country Link
CN (1) CN111971389A (fr)
WO (1) WO2019184655A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024168092A3 (fr) * 2023-02-07 2024-09-26 Parse Biosciences, Inc. Procédés et kits de marquage de molécules cellulaires pour analyse multiplex
WO2025217892A1 (fr) * 2024-04-19 2025-10-23 深圳市先康达生命科学有限公司 Procédé d'édition génique pour le knock-in simultanée de fragments d'adn au niveau de plusieurs sites et application associée

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118325866B (zh) * 2024-03-01 2025-05-27 上海交通大学 一种基因编辑系统在制备基因编辑产品中的用途
CN120866325A (zh) * 2025-09-28 2025-10-31 隆平生物技术(海南)有限公司 一种提高基因编辑效率的高效sgRNA及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015138855A1 (fr) * 2014-03-14 2015-09-17 The Regents Of The University Of California Vecteurs et méthodes d'ingénierie génomique fongique à l'aide de crispr-cas9
CN105255937A (zh) * 2015-08-14 2016-01-20 西北农林科技大学 一种真核细胞III型启动子表达CRISPR sgRNA的方法及其应用
CN106520824A (zh) * 2016-09-30 2017-03-22 北京大北农科技集团股份有限公司 多靶点编辑系统及其用途
CN107475256A (zh) * 2017-08-01 2017-12-15 西南大学 一种基于内源tRNA加工系统的多靶序列sgRNA表达载体及其在植物基因编辑中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160130392A (ko) * 2014-02-18 2016-11-11 듀크 유니버시티 바이러스 복제의 불활성화를 위한 조성물 및 그의 제조 및 사용 방법
EP3207139B1 (fr) * 2014-10-17 2025-05-07 The Penn State Research Foundation Procédés et compositions pour l'édition génomique multiplex à guidage arn et autres technologies d'arn

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015138855A1 (fr) * 2014-03-14 2015-09-17 The Regents Of The University Of California Vecteurs et méthodes d'ingénierie génomique fongique à l'aide de crispr-cas9
CN105255937A (zh) * 2015-08-14 2016-01-20 西北农林科技大学 一种真核细胞III型启动子表达CRISPR sgRNA的方法及其应用
CN106520824A (zh) * 2016-09-30 2017-03-22 北京大北农科技集团股份有限公司 多靶点编辑系统及其用途
CN107475256A (zh) * 2017-08-01 2017-12-15 西南大学 一种基于内源tRNA加工系统的多靶序列sgRNA表达载体及其在植物基因编辑中的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MA, CUNFA: "Preliminary Studies on Gene Editing in Brassica Oleracea L. Using CRISPR/Cas9 System", CHINESE MASTER'S THESES FULL-TEXT DATABASE (ELECTRONIC JOURNAL, 15 February 2018 (2018-02-15), ISSN: 1674-0246 *
NUMAMOTO, M ET AL.: "Efficient Genome Editing by CRISPR/Cas9 with a Trna-Sgrna Fusion the Methylotrophic Yeast & Itogataea Polymorpha", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 124, no. 5, 31 December 2017 (2017-12-31), pages 487 - 492, XP085237843, ISSN: 1389-1723, DOI: 10.1016/j.jbiosc.2017.06.001 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024168092A3 (fr) * 2023-02-07 2024-09-26 Parse Biosciences, Inc. Procédés et kits de marquage de molécules cellulaires pour analyse multiplex
WO2025217892A1 (fr) * 2024-04-19 2025-10-23 深圳市先康达生命科学有限公司 Procédé d'édition génique pour le knock-in simultanée de fragments d'adn au niveau de plusieurs sites et application associée

Also Published As

Publication number Publication date
CN111971389A (zh) 2020-11-20

Similar Documents

Publication Publication Date Title
US20220033858A1 (en) Crispr oligoncleotides and gene editing
CN106047877B (zh) 一种靶向敲除FTO基因的sgRNA及CRISPR/Cas9慢病毒系统与应用
US20190264193A1 (en) Protein engineering methods
CN109136248B (zh) 多靶点编辑载体及其构建方法和应用
CN113939591A (zh) 编辑rna的方法和组合物
CN116218836A (zh) 编辑rna的方法和组合物
EP4491723A1 (fr) Cytosine désaminase et son utilisation dans l'édition de bases
CN110835632B (zh) 新型碱基转换编辑系统用于基因治疗的应用
WO2019184655A1 (fr) Application de système crispr/cas dans l'édition de gènes
CN103388006A (zh) 一种基因定点突变的构建方法
CN109415729A (zh) 具有降低毒性的基因编辑试剂
US20210130817A1 (en) Gene Editing System and Gene Editing Method
CN109706148A (zh) 一种用于敲除BCL11A基因或者BCL11A基因增强子的gRNA、gRNA组合物以及电转方法
WO2021175289A1 (fr) Procédé et système d'édition de génome multiplex
WO1999027072A1 (fr) Reactifs et procedes pour la diversification d'adn
CN111304172A (zh) 一种基于CRISPR-Cas9编辑技术的敲除鸡EphA2基因的细胞系的构建方法
JP7606176B2 (ja) ゲノム編集効率を向上させる融合タンパク質及びその使用
CN114085841A (zh) 一种cho细胞基因nw_003614092.1内稳定表达蛋白质的位点及其应用
CN117448324A (zh) 基于CRISPR/Cas9系统高效编辑水牛H11位点的gRNA
CN113969284B (zh) 一种cho细胞基因nw_003614889.1内稳定表达蛋白质的位点及其应用
JP7026304B2 (ja) 部位特異的ｄｎａ開裂及び修復による標的化原位置タンパク質多様化
KR102421129B1 (ko) 신규 프로토스페이서 인접 모티프 서열 및 이를 이용한 세포의 유전체에서 표적 핵산을 변형시키는 방법
WO2023141734A1 (fr) Système d'édition primaire modifié et son utilisation
CN113969283A (zh) 一种cho细胞基因nw_003613756.1内稳定表达蛋白质的位点及其应用
KR102638799B1 (ko) CRISPR/Cas12f1(Cas14a1) system 효율화를 위한 engineered guide RNA 및 이의 용도

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19777972

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19777972

Country of ref document: EP

Kind code of ref document: A1