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WO2019127116A1 - Clone cellulaire pour études phénotypiques cellulaires, procédé de criblage associé et application correspondante - Google Patents

Clone cellulaire pour études phénotypiques cellulaires, procédé de criblage associé et application correspondante Download PDF

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WO2019127116A1
WO2019127116A1 PCT/CN2017/119054 CN2017119054W WO2019127116A1 WO 2019127116 A1 WO2019127116 A1 WO 2019127116A1 CN 2017119054 W CN2017119054 W CN 2017119054W WO 2019127116 A1 WO2019127116 A1 WO 2019127116A1
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grna
cell
barcode
vector
screening
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蓝田
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Yunzhou Biosciences (guangzhou) Inc
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Yunzhou Biosciences (guangzhou) Inc
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    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid

Definitions

  • the invention relates to the field of molecular biology, in particular to a cell clone for cell phenotype research and a screening method and application thereof.
  • tumor cell populations will exhibit different phenotypes, such as tumor cells that can proliferate, some migrate, and some have drug resistance. These different phenotypes are progress in cancer research. There are many major causes of unpredictability in response to treatment, so understanding the basic biology of tumorigenesis is very important to improve treatment outcomes.
  • the underlying cause of tumor cells with different phenotypes can be attributed to two mutually non-exclusive possibilities: one is that all tumor cells in the patient have the same intrinsic phenotypic potential, but there are many factor variables in the patient's body itself or during the medication.
  • the search tool used is a lentiviral shRNA interference vector, which specifically targets the reporter gene on the barcode tool. Fluorescent reporter cells are weakened by the target cells.
  • a screening method for cell clones for cell phenotype research comprising the following steps:
  • Step 1 construct a gRNA barcode vector having an expression cassette for expressing gRNA used as a marker barcode, the gRNA barcode vector has various types, and different gRNA barcode vectors have different sequences of gRNA coding fragments, and various
  • the gRNA barcode carrier constitutes a gRNA barcode carrier;
  • Step 2 transducing the gRNA barcode carrier library to the cells to be studied, and obtaining a cell population composed of a plurality of cells containing different gRNA coding fragments, and dividing the cell population into an experimental group and a reserved group;
  • Step 3 experimentally treating the cell population of the experimental group, screening the experimental group positive cells having a specific phenotype, and obtaining the sequence of the specific gRNA coding fragment corresponding to the specific phenotype in the positive cells of the experimental group. ;
  • Step 4 constructing a specific gRNA-sensor search vector for the sequence of the specific gRNA-encoding fragment obtained, the expression frame of the gRNA-sensor search vector containing the first reporter gene and inserted in the first reporter gene a left homology arm, a specific gRNA coding fragment, a PAM and a right homology arm, the left homology arm and the right homology arm being homologous to a partial sequence fragment of the first reporter gene;
  • Step 5 After the constructed specific gRNA-sensor search vector and the vector capable of expressing the nuclease for binding the gRNA barcode are used to transduce the cell population of the reserved group, the result is selected according to the expression of the first reporter gene. Positive cells, that is.
  • the constructing the gRNA barcode vector is to insert the gRNA-encoding fragment into a vector having a suicide gene to replace the suicide gene.
  • the gRNA-encoding fragment is further ligated to the protective sequence fragment and/or the cleavage site recognition fragment, respectively.
  • the gRNA barcode has a length of 18-23 bp, preferably 20 bp.
  • the protected sequence fragment is 40-50 bp in length, preferably 40 bp.
  • the gRNA barcode vector further has an expression cassette for a second reporter gene.
  • the second reporter gene and/or the first reporter gene is a fluorescent protein gene and/or a drug resistance gene.
  • the gRNA barcode vector is a lentiviral vector.
  • each cell to be studied is transduced with one or two gRNA barcode carriers.
  • the step of expanding the resulting population of cells is further included prior to dividing the population of cells into an experimental group and a reserved group.
  • the left homology arm and/or the right homology arm are 180-300 bp in length, preferably 200 bp.
  • the vector capable of expressing a nuclease for binding to a gRNA barcode is a vector capable of expressing a Cas9 or saCas9 nuclease.
  • the above cell clones are used in the study of cell phenotypes or in the preparation of reagents for studying cell phenotypes.
  • the screening method for cell clones for cell phenotypic research of the present invention can be mainly used for searching for a cell phenotype (including a tendency to respond to an experimental treatment) from a cell that has not been subjected to experimental treatment, wherein gRNA
  • the barcode carrier acts both as a barcode and as a retrieval tool, thus simplifying the process of searching and screening.
  • the first reporter gene of the gRNA-sensor search vector itself is not normally expressed, only with the specific gRNA barcode and The signal is signaled by binding to the nuclease of the gRNA barcode, so that the search process can be characterized; again, as long as there is a signal related to the first reporter gene, it can be captured, thereby facilitating screening to obtain positive cell clones.
  • Screened positive cells can be used in a single study to determine their ability to respond or phenotype to further determine the underlying cause of cells with different phenotypes, and can be widely used in studying cell phenotypes or in preparing for studying cell phenotypes. In the reagents.
  • Figure 1 is a schematic diagram showing the screening process of cell clones for cell phenotype research
  • NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN is a gRNA coding fragment of a gRNA barcode
  • Figure 3 is a schematic diagram showing the structure of a gRNA-sensor search vector.
  • CRISPR/Cas9 (or variants thereof, such as saCas9) (Clustered Regularly Interspaced Short Palindromic Repeats) is a technique by which RNA directs Cas nuclease to perform specific DNA modification of a targeted gene.
  • gRNA binds to nucleases such as Cas9 and saCas9
  • the nuclease cleaves the DNA duplex by a PAM (Protospacer Adjacent Motif, NGG) sequence, resulting in DNA double-strand break.
  • PAM Protospacer Adjacent Motif, NGG
  • a screening method for cell clones for cell phenotypic research of an embodiment is mainly used for constructing a nuclease-assisted search for barcode cell clones, and screening for positive cell clones having a certain phenotype (including a tendency to respond to experimental treatment), Cell phenotypes were studied in depth.
  • the screening method of this embodiment includes the following steps:
  • Step 1 constructing a gRNA barcode vector having an expression cassette for expressing gRNA used as a marker barcode, the gRNA barcode vector is various, and different gRNA barcode vectors have different sequences of gRNA coding fragments, and various gRNAs
  • the barcode carrier constitutes a library of gRNA barcode carriers.
  • the gRNA barcode is a fragment of a gRNA sequence having a specific sequence.
  • each gRNA barcode vector and a cell subsequently transduced with the gRNA barcode vector have a specific gene signature.
  • the length of the gRNA barcode is 18-23 bp, preferably 20 bp (the sequence of the gRNA barcode of 20 bp length can be expressed as "NNNNNNNNNNNNNNNNNNNNNNNN", that is, there are 4 20 kinds of gRNA barcodes theoretically having the length. Different combinations.
  • the gRNA barcode vector has at least one expression cassette for expressing a gRNA barcode, and the expression cassette is composed of a gRNA coding fragment of a gRNA barcode and a corresponding promoter, terminator and the like.
  • the gRNA-encoding fragment for expression of the gRNA barcode is further ligated with a protective sequence fragment and/or a restriction endonuclease fragment fragment, respectively.
  • the protected sequence fragments and/or restriction endonuclease site fragments can be used as universal amplification primer fragments for different gRNA barcodes for PCR amplification and/or restriction endonuclease digestion.
  • the length of the protected sequence fragment can be, but is not limited to, 40-50 bp, preferably 40 bp.
  • the empty vector used to construct the gRNA barcode vector has a suicide gene, such as a ccdB gene, into which the gRNA-encoding fragment of the gRNA barcode is inserted.
  • the suicide gene can reduce the effect of the vector without the target gene, and the bacteria that subsequently select for transfection also use bacteria that cannot resist the suicide gene.
  • the gRNA barcode vector further comprises another expression cassette for expression of a second reporter gene.
  • the second reporter gene can be, but is not limited to, a fluorescent protein gene and/or a drug resistance gene.
  • the design of the second reporter group facilitates the labeling and screening of vectors into which the sequence of interest is inserted.
  • the gRNA barcode vector is a lentiviral vector
  • the promoter of the gRNA coding fragment containing the gRNA barcode is a U6 promoter
  • the gRNA barcode of the gRNA barcode is inserted in U6 Downstream of the promoter, replacing the original suicide ccdB gene fragment
  • the promoter of another expression cassette is the PGK promoter
  • the second reporter gene is a fluorescent protein gene such as mCherry and a drug resistance gene such as Puro, wherein the fluorescent protein gene can be used for expression.
  • the fluorescently-labeled fluorescent protein, the drug-resistant gene makes the vector or the transfected cell resistant, and can be used for drug screening and enrichment of the desired expression vector or cell.
  • the gRNA barcode vector can be, but is not limited to, a lentiviral vector
  • the promoter of the gRNA barcode can be U6 but not limited to the U6 promoter
  • the promoter of the reporter gene expression cassette can be, but is not limited to, a PGK promoter.
  • Step 2 The gRNA barcode carrier library is transduced into the cells to be studied, and a cell population composed of a plurality of cells containing different gRNA coding fragments is obtained, and the cell population is divided into an experimental group and a reserved group.
  • the amount of vector used in the gRNA barcode vector library is adjusted such that each cell to be studied is randomly transduced with one or two gRNA barcode carriers.
  • the step of expanding the obtained cell population is further included before dividing the cell population into the experimental group and the reserved group.
  • the obtained experimental group cell group and the reserved group cell group have substantially the same number of cells labeled with each gRNA barcode, and have multiple copies, such as expanded culture, so that each gRNA barcode-labeled cell has about 100 sisters. Cells for easy analysis.
  • Step 3 Experimentally treating the cell population of the experimental group, screening the experimental group positive cells having a specific phenotype, and obtaining the sequence of the specific gRNA coding fragment corresponding to the specific phenotype in the positive cells of the experimental group.
  • sequence of the specific gRNA-encoding fragment corresponding to the gRNA barcode in the experimental group positive cells can be separately read by, but not limited to, PCR or sequencing.
  • Step 4 construct a specific gRNA-sensor search vector for the sequence of the specific gRNA-encoding fragment obtained, and the gRNA-sensor search vector contains the first reporter gene and the left homolog inserted in the first reporter gene.
  • the arm, the specific gRNA coding fragment, the PAM and the right homology arm, the left homologous arm and the right homology arm are homologous to a partial sequence fragment of the first reporter gene.
  • the left homology arm and/or the right homology arm are 180-300 bp in length, preferably 200 bp.
  • the promoter of the gRNA-sensor search vector may be EF1A
  • the first reporter gene may be a green fluorescent protein (EGFP) gene or the like.
  • EGFP green fluorescent protein
  • Step 5 After the constructed specific gRNA-sensor search vector and the vector capable of expressing the nuclease for binding the gRNA barcode are used to transduce the cell population of the reserved group, the positive cells are selected according to the expression of the first reporter gene. That's it.
  • the vector capable of expressing a nuclease for binding to a gRNA barcode is a vector capable of expressing a nuclease such as Cas9 or saCas9.
  • the nucleotide of the first reporter gene (such as EGFP gene) is divided into two parts, the upstream part and the downstream part, by the gRNA coding fragment + PAM of the specific gRNA barcode. There are left and right homology arms of about 200 bp in length. At this time, the first reporter gene cannot be expressed normally.
  • the specific gRNA-sensor search vector is transferred to the cell group of the reserved group together with the vector capable of expressing the nuclease for binding the gRNA barcode, the experimental group positive cells selected in the above step 3 have the same gRNA barcode.
  • the gRNA expressed in the cell can be combined with the nuclease, and the target-specific gRNA-sensor search vector can be searched. At this time, the left and right homologous arms of the first reporter gene on the gRNA-sensor search vector are homologously recombined, first
  • the reporter gene is normally expressed, such as the EGFP gene expressing EGFP, which emits a green fluorescent signal for screening.
  • Positive cell clones can be screened from the reserved group by signal changes in the expression of the first reporter gene from scratch, such as positive cell clones that can be screened for specific fluorescent signals by flow cytometry, and The positive cell clones with specific drug resistance are screened by drugs, and the selected positive cell clones are separately expanded and cultured, which can be used to further study the ability of response or phenotype formation, and the cells have different phenotypes. s reason. Therefore, the cell clones screened by the above screening methods can be used in the study of cell phenotypes or in the preparation of reagents for studying cell phenotypes.
  • the screening method of the cell clone for the above cell phenotypic study can be mainly used for searching a cell population of a certain cell phenotype (including a tendency to respond to an experimental treatment) from the untreated cells, wherein the gRNA barcode carrier It acts both as a barcode and as a search tool, thus simplifying the process of searching and screening.
  • the first reporter gene of the gRNA-sensor search vector itself is not normally expressed, only with specific gRNA barcodes and for binding to gRNA.
  • the nuclease of the barcode signals the signal, so that the process can be characterized qualitatively; again, as long as there is a signal related to the first reporter gene, it can be captured, thereby facilitating screening to obtain positive cell clones.
  • mouse breast cancer cell TM40D-MB can be transferred to bone and form secondary tumors after implantation into mouse mammary fat pad.
  • This cell line is a series of continuous cells in BALB/c mice by TM40 mother cells. Obtained from the transplant experiment.
  • the Luc luciferase reporter gene was integrated into TM40D-MB (Li, Z., C. Schem, YHShi, D. Medina, and M. Zhang, Increased COX2 expression enhances tumor- In induced osteoclastic lesions in breast cancer bone metastasis. Clin Exp Metastasis, 2008. 25(4): p.
  • TM40D-MB-Luc cells were formed.
  • the ability of metastatic tumors to metastasize may be caused by random causes or by genetic factors. Studies have shown that cells derived from secondary tumors have stronger transfer ability, and it is never necessary to find out the root cause of their stronger metastatic potential. This method has never used the method of "nuclease-assisted search for barcode cloning".
  • Sister cells that can form secondary tumors are obtained from transplanted mouse breast cancer cells TM40D-MB-Luc, and whether the formation of secondary tumors is random or whether these cells themselves have greater metastatic potential, the specific steps are as follows.
  • the expression cassette 1 is the U6 promoter-mediated expression of the gRNA-encoding fragment of gRNA; the expression cassette 2 drives the red fluorescent protein mCherry and puromycin Puro drug screening.
  • the gene, red fluorescent protein is a visible marker, so that the gRNA barcode carrier can be observed after entering the cell, and puromycin can eliminate cells that do not contain the gRNA barcode vector.
  • each gRNA coding fragment is used to encode a gRNA barcode of 20 nt in length, and an additional amplification region is provided at both ends of the gRNA coding fragment,
  • the amplification region comprises an enzyme cleavage site and a protective base (40 bases at both ends) for cloning the gRNA coding fragment;
  • sequences of partial gRNAs in the gRNA-encoding fragment library used are as follows:
  • gRNA1 TACGTCCCTGTGCAGCTGCA (SEQ ID NO. 1)
  • gRNA2 ACGTCCCTGTGCAGCTGCAA (SEQ ID NO. 2)
  • gRNA3 CGTCCCTGTGCAGCTGCAAG (SEQ ID NO. 3)
  • gRNA4 TACGTCCCTGTGCAGCTGCA (SEQ ID NO. 4)
  • gRNA5 GCACTACCAGAGCTAACTCA (SEQ ID NO. 5)
  • gRNA6 GGGGCCACTAGGGACAGGAT (SEQ ID NO. 6)
  • the primers for PCR amplification are as follows:
  • the gRNA barcode vector is packaged into a lentiviral vector, and the amount of virus is adjusted according to the number of TM40D-MB-Luc cells, so that each cell can randomly contain one or two gRNA barcodes.
  • the red fluorescent protein mCherry was used to observe the virus transduction quality, and the puromycin Puro was used to screen the clones that obtained the gRNA barcode, and the cells containing the gRNA barcode library were expanded to be 6-7 generations so that each cell could contain about 100 sisters. cell.
  • the amplified cells are divided into two parts, one as an experimental group and one as a reserved group.
  • the experimental group was subjected to the following treatment: 2 ⁇ 10 7 TM40D-MB-Luc cells were transplanted into 20 mouse breast tissues of BALB/c, and 1 ⁇ 10 6 cells were transplanted per mouse.
  • the expression of luciferase Luc was observed using an optical microscopy imaging system, and the size of the tumor was monitored three times per week, and it was found that TM40D-MB-Luc cells formed a primary tumor of about 0.2 cm in the breast.
  • half of the mice showed tumor metastasis to the bones, and the secondary tumors on the bones were monitored weekly to record the size and number of secondary tumors and the life span of the mice.
  • each secondary tumor cell is developed from a cell with metastatic potential.
  • Each secondary tumor cell is isolated and expanded separately, genomic DNA is extracted, and the gRNA coding fragment is amplified by PCR amplification. The coding sequence of a specific gRNA barcode is confirmed by sequencing.
  • the gRNA+PAM sequence fragment is cloned into the middle of the green fluorescent protein EGFP according to the sequence of the specific gRNA barcode, and specifically includes the following steps:
  • gRNA1-F ctaaTACGTCCCTGTGCAGCTGCAAGG (SEQ ID NO. 13)
  • gRNA1-R cgggCCTTGCAGCTGCACAGGGACGTA (SEQ ID NO. 14)
  • gRNA2-F ctaaACGTCCCTGTGCAGCTGCAAGGG (SEQ ID NO. 15)
  • gRNA2-R cgggCCCTTGCAGCTGCACAGGGACGT (SEQ ID NO. 16)
  • the promoter plasmid such as pUp-EF1A
  • the reporter plasmid pDown-DR-EGFP-gRNA-PAM
  • the target plasmid pRp
  • the specific gRNA-sensor vector and the vector expressing Cas9 protein were separately transduced into the reserved cells, and the cell clones with green fluorescent signals were screened by flow cytometry (FACS). These clones have metastatic potential. Sisters were positive for barcode cells and some clones without fluorescent signal were screened as experimental negative controls.
  • Each sister-positive barcode cell population was transplanted into the breast tissue of 20 BALB/c mice with the same number (1 ⁇ 10 6 cells), and the size of the primary tumor and the secondary tumor were recorded. Data on the number and size of mice, the lifespan of mice, and the like, and cells with no metastatic ability (no fluorescence) were used as negative controls. These data were compared to experimental group data to analyze whether these cells have greater metastatic potential.
  • metastatic ability is caused by random causes; if the primary tumor appears faster, larger, and transferred to the bone The more or faster the next generation of tumors, it can be considered that this metastatic ability is determined by genetic factors, and thus can carry out genotypic analysis of molecular biology such as whole genome sequencing, DNA methylation analysis.

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Abstract

La présente invention concerne un clone cellulaire pour des études phénotypiques cellulaires, un procédé de criblage associé et une application correspondante. Le procédé de criblage pour le clone cellulaire pour des études phénotypiques cellulaires est principalement applicable lors de la recherche d'une population cellulaire d'un certain phénotype cellulaire dans des cellules n'ayant pas été soumises à un traitement expérimental. Un vecteur de code-barres d'ARNg agit à la fois comme code-barres et comme outil de récupération, ce qui simplifie un procédé de recherche et de criblage. Un premier gène rapporteur d'un vecteur de recherche de capteur d'ARNg lui-même n'est pas normalement exprimé et signale uniquement en réponse à une action avec un code-barres d'ARNg spécifique et une nucléase pour se lier au code-barres d'ARNg, permettant ainsi la qualification dans le procédé de recherche. Tant qu'un signal associé au premier gène rapporteur est présent, le signal peut être capturé, ce qui facilite le criblage pour obtenir des clones cellulaires positifs. Les cellules positives sélectionnées peuvent être utilisées dans des études uniques et peuvent répondre à ou générer des phénotypes. Les cellules sélectionnées peuvent également être largement utilisées dans l'étude de phénotypes cellulaires ou dans la préparation de réactifs pour l'étude de phénotypes cellulaires.
PCT/CN2017/119054 2017-12-25 2017-12-27 Clone cellulaire pour études phénotypiques cellulaires, procédé de criblage associé et application correspondante Ceased WO2019127116A1 (fr)

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EP3912644A4 (fr) * 2019-01-18 2022-09-28 Osaka University Agent thérapeutique pour l'épidermolyse bulleuse dystrophique
CN117625619B (zh) * 2023-12-05 2024-11-12 云舟生物科技(广州)股份有限公司 核酸分子及其作为特异启动子的应用

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CN102203281A (zh) * 2009-08-21 2011-09-28 赛业（广州）生物科技有限公司 复制条码筛选检测
WO2016174151A1 (fr) * 2015-04-30 2016-11-03 Roche Diagnostics Gmbh Détection et détermination de phénotype de manière spécifique de séquence
WO2016196361A1 (fr) * 2015-05-29 2016-12-08 North Carolina State University Procédés pour le criblage de bactéries, d'archées, d'algues et de levure à l'aide d'acides nucléiques crispr

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ES2844174T3 (es) * 2013-09-18 2021-07-21 Kymab Ltd Métodos, células y organismos
PL3152312T3 (pl) * 2014-06-06 2020-08-10 Regeneron Pharmaceuticals, Inc. Sposoby i kompozycje do modyfikowania docelowego locus

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Publication number Priority date Publication date Assignee Title
CN102203281A (zh) * 2009-08-21 2011-09-28 赛业（广州）生物科技有限公司 复制条码筛选检测
WO2016174151A1 (fr) * 2015-04-30 2016-11-03 Roche Diagnostics Gmbh Détection et détermination de phénotype de manière spécifique de séquence
WO2016196361A1 (fr) * 2015-05-29 2016-12-08 North Carolina State University Procédés pour le criblage de bactéries, d'archées, d'algues et de levure à l'aide d'acides nucléiques crispr

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