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WO2021089984A1 - Identification médiée par crispr de protéines biotinylées et de régions de chromatine - Google Patents

Identification médiée par crispr de protéines biotinylées et de régions de chromatine Download PDF

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WO2021089984A1
WO2021089984A1 PCT/GB2020/052596 GB2020052596W WO2021089984A1 WO 2021089984 A1 WO2021089984 A1 WO 2021089984A1 GB 2020052596 W GB2020052596 W GB 2020052596W WO 2021089984 A1 WO2021089984 A1 WO 2021089984A1
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Prior art keywords
protein
seq
cas9
acid sequence
biotin
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Metodi METODIEV
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University of Essex Enterprises Ltd
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University of Essex Enterprises Ltd
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/04—Other carbon-nitrogen ligases (6.3.4)
    • C12Y603/04015—Biotin-[acetyl-CoA-carboxylase] ligase (6.3.4.15)
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • 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
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR-Cas Clustered Regularly Interspaced Short Palindromic Repeats
  • RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. This RNA, which contains sequence complementary to the viral genome, mediates targeting of an RNA- guided nuclease to a target sequence in the viral genome. The RNA-guided nuclease, in turn, cleaves and thereby silences the viral target.
  • Suitable dCas domains can be obtained from a Cas system.
  • the Cas can be a type I, a type II or a type III system.
  • Non-limiting examples of suitable dCas domains can be from Cas1 , Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8 and Cas10, for example.
  • a particularly suitable dCas domain can be a dCas9.
  • the dCas9 can be obtained, for example, by introducing point mutations and/or deletions in the Cas9 protein at both the RuvC and HNH protein active sites (see, Jinek et al., Science 2012; 337:816-821).
  • the fusion protein and system described herein comprise a proximity dependent protein.
  • the term “proximity-dependent” may refer to nucleic acid molecules or proteins within about 10nm of the fusion proteins disclosed herein.
  • the BirA enzyme is a mutant BirA enzyme derived from Aquifex aeolicus.
  • the proximity dependent protein is a mutant enzyme derived from Aquifex aeolicus and described in Kim et ai ⁇ Mol. Biol. Cell. 2016 Feb 24 p1188- 1196 (referred to as BiolD2).
  • the NLS is fused at the 3’ end of the nucleic acid molecule encoding the nucleotide binding protein (e.g. dCas) coding sequence.
  • a NLS e.g. SV40 NLS is also fused to a 5’ end of the nucleic acid molecule encoding the nucleotide binding protein.
  • gRNAs comprise two separate RNA molecules: an "activator- RNA” and a “targeter-RNA.”
  • Other gRNAs are a single RNA molecule (single RNA polynucleotide), which can also be called a “single-molecule gRNA,” a “single-guide RNA,” or an "sgRNA”.
  • the system comprises a plurality of gRNA, each gRNA being specific to a region of a gene of interest.
  • the nucleic acid molecule encoding the fusion protein further comprises one or more promoter sequences.
  • the promoter driving expression of the fusion protein is a constitutive promoter such as a cytomegalovirus (CMV) promoter.
  • the nucleic acid molecule comprises an inducible expression system such as for example a TetO inducible expression system (TetO) (see Reference 3 for details of implementing the TetO system).
  • the promoter is a Tet-On promoter.
  • the system work on the principle of “inhibiting the inhibitor”. In the absence of tetracycline a repressor protein binds to the promoter and blocks transcription of the gene. In the presence of tetracycline the repressor is inactivated and transcription occurs.
  • the two constructs one, referred to as TetR drives the constitutive expression of the repressor protein.
  • the other is the gene construct, which contains the tetracycline-responsible promoter.
  • TetR drives the constitutive expression of the repressor protein.
  • the other is the gene construct, which contains the tetracycline-responsible promoter.
  • these two constructs are integrated in the genome and stable cell lines are produced.
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.”
  • Expression vectors in recombinant DNA techniques often take the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • next generation gene sequencing refers to a DNA sequencing technology which is able to sequence large amount of genetic material.
  • NGS platforms There are a number of different NGS platforms which may be used in the present invention.
  • the resulting DNA product was digested with BamHI and EcoRI and gel-purified.
  • the VP64 coding sequence was excised from pAC152-dual-dCas9VP64 with BamHI and EcoRI and the linearized plasmid was gel-purified.
  • oligonucleotides were designed for targeting 6 different loci in the promoter of human STAT1 :
  • Cells were transfected and labelled in 10cm plates. Transfected cells were washed twice with cold PBS. Lysis buffer (10mM Tris-CI pH 7.5, 1% Igepal Ca-640, 2M Urea, phosphatase and protease inhibitors) was added (0.5ml per plate) and the cells were scrapped off the plate and transferred to a clean Eppendorf tube. The tubes were incubated on a rotator at room temperature (RT) rotating for 15min to allow complete lysis. The cell lysates were then centrifuged in a table-top refrigerated centrifuge at 10,000g for 15min. The clear supernatants were transferred to a clean low-binding microfuge tube (to minimize plastic contamination).
  • Lysis buffer (10mM Tris-CI pH 7.5, 1% Igepal Ca-640, 2M Urea, phosphatase and protease inhibitors) was added (0.5ml per plate) and the cells were scrapped off the plate and transferred to a clean
  • the beads were pelleted by centrifugation at 1000g for 1 min and the supernatant transferred to a separate clean tube to be kept as flow-through sample (FT).
  • the beads were washed 3 times with 1 ml lysis buffer without protease and phosphatase inhibitors. The beads were then washed once with 1 ml PBS. All liquid was aspirated using gel-loading tips and 20 ⁇ I of trypsin solution (sequencing-grade trypsin from Promega, 20ng/ ⁇ I), was added. Beads were incubated overnight at 30°C.
  • the peptide samples were aspirated from the digests using gelloading tips and 5 ⁇ I formic acid (20% v/v) were added to stop digestion and acidify the samples. The acidified peptide samples were kept at -80°C until analysed by LC-MS/MS.
  • gRNA The expression of gRNA is driven by the U6 promoter which acts via Pol III ensuring efficient expression of shRNA molecules.
  • APLSASMIK (SEQ. ID. No. 20) 916.50519 HA-Cas9 4.08E-05 158.07 AQVSGQGDSLHEHIANLAGSPAIK (SEQ. ID. 2399.2193 HA-Cas9 6.55E-10 169.6 No. 21)
  • EIFFDQSK (SEQ. ID. No. 25) 1012.4866 HA-Cas9 0.0020923 122.18
  • ELGSQILK (SEQ. ID. No. 27) 886.51238 HA-Cas9 0.00086045 143.12
  • EQAENIIHLFTLTNLGAPAAFK SEQ. ID. No. 2397.2692 HA-Cas9 3.59E-11 180.27 29
  • KIECFDSVEISGVEDR (SEQ. ID. No. 52) 1881.8778 HA-Cas9 4.50E-13 222.88
  • KLVDSTDKADLR (SEQ. ID. No. 53) 1359.7358 HA-Cas9 1.42E-05 164.48
  • KNGLFGNLIALSLGLTPNFK (SEQ. ID. No. 54) 2116.2045 HA-Cas9 0.044953 47.729
  • KPAFLSGEQK (SEQ. ID. No. 56) 1103.5975 HA-Cas9 0.00019886 154.01
  • KPAFLSGEQKK (SEQ. ID. No. 57) 1231.6925 HA-Cas9 0.023537 87.713
  • KSEETITPWNFEEVVDK (SEQ. ID. No. 59) 2049.9895 HA-Cas9 2.33E-06 157.84
  • KSEETITPWNFEEVVDKGASAQSFIER (SEQ. 3096.504 HA-Cas9 1.36E-16 176.23
  • KVLSMPQVNIVK (SEQ. ID. No. 62) 1354.8006 HA-Cas9 1.93E-15 199.8
  • LEESFLVEEDKKHER (SEQ. ID. No. 66) 1886.9374 HA-Cas9 6.87E-38 232.78
  • LENLIAQLPGEK (SEQ. ID. No. 67) 1323.7398 HA-Cas9 6.26E-05 142.89
  • LENLIAQLPGEKK (SEQ. ID. No. 68) 1451.8348 HA-Cas9 0.0054437 88.163
  • LIYLALAHMIK (SEQ. ID. No. 70) 1284.7628 HA-Cas9 7.97E-07 163.67
  • LKTYAHLFDDK (SEQ. ID. No. 72) 1349.698 HA-Cas9 0.0015331 107.11
  • LSDYDVDAIVPQSFLK (SEQ. ID. No. 73) 1808.9196 HA-Cas9 8.07E-07 206.32
  • LVDSTDKADLR (SEQ. ID. No. 75) 1231.6408 HA-Cas9 0.011106 80.24 LYLYYLQNGR (SEQ. ID. No. 76) 1301.6768 HA-Cas9 1.08E-25 231.86 MDGTEELLVK (SEQ. ID. No. 77) 1133.5638 HA-Cas9 3.69E-19 226.47 MLASAGELQK (SEQ. ID. No. 78) 1046.543 HA-Cas9 1.41E-18 218.73
  • MTNFDKNLPNEK (SEQ. ID. No. 79) 1449.6922 HA-Cas9 8.65E-14 193.62
  • MYPYDVPDYASPK (SEQ. ID. No. 80) 1544.6857 HA-Cas9 7.65E-05 136.49
  • NFMQLIHDDSLTFK (SEQ. ID. No. 81) 1707.829 HA-Cas9 0.0046976 82.705
  • NLIGALLFDSGETAEATR SEQ. ID. No. 84
  • NLSDAILLSDILR (SEQ. ID. No. 85) 1441.814 HA-Cas9 3.35E-96 327.1
  • NSDKLIAR (SEQ. ID. No. 87) 915.51378 HA-Cas9 0.014864 146.58
  • RIEEGIKELGSQILK (SEQ. ID. No. 90) 1711.9832 HA-Cas9 0.016102 80.411
  • RLENLIAQLPGEK (SEQ. ID. No. 91) 1479.8409 HA-Cas9 1.82E-05 158.34
  • RQEDFYPFLK (SEQ. ID. No. 94) 1341.6717 HA-Cas9 0.0034627 139.38
  • RQEDFYPFLKDNR (SEQ. ID. No. 95) 1726.8427 HA-Cas9 0.026613 75.764
  • RVILADANLDK (SEQ. ID. No. 96) 1226.6983 HA-Cas9 0.0014377 134.75
  • SDNVPSEEVVK (SEQ. ID. No. 98) 1201.5826 HA-Cas9 0.0007638 115.7
  • SNFDLAEDAK (SEQ. ID. No. 102) 1108.5037 HA-Cas9 9.37E-09 179.42
  • TEITLANGEIR SEQ. ID. No. 104 1215.6459 HA-Cas9 1.39E-41 266.17
  • TEVQTGGFSK (SEQ. ID. No. 105) 1052.5138 HA-Cas9 6.63E-06 159.34
  • TILDFLK (SEQ. ID. No. 107) 848.50076 HA-Cas9 0.011582 104.45
  • VDDSFFHR (SEQ. ID. No. 108) 1021.4617 HA-Cas9 0.0012042 134.81
  • VDDSFFHRLEESFLVEEDKK (SEQ. ID. No. 2468.186 HA-Cas9 0.0027018 74.793 109)
  • VILADANLDKVLSAYNK (SEQ. ID. No. 111) 1846.02 HA-Cas9 0.006281 93.51
  • VLSMPQVNIVK (SEQ. ID. No. 112) 1226.7057 HA-Cas9 3.77E-10 182.39
  • YFFYSNIMNFFK (SEQ. ID. No. 117) 1619.7483 HA-Cas9 5.79E-07 163.66
  • YGG FDSPTVAYSVLVVAK (SEQ. ID. No. 118) 1871.9669 HA-Cas9 6.88E-84 320.07
  • YPTIYHLR (SEQ. ID. No 120) 1061.5658 HA-Cas9 0.00056907 150.17
  • YVNFLYLASHYEK (SEQ. ID. No. 122) 1645.814 HA-Cas9 1.22E-21 219.92
  • YVTEGMR (SEQ. ID. No. 123) 854.39564 HA-Cas9 0.0031948 115.46
  • EILSGEFSLR SEQ. ID. No. 127) 1149.603 Bio-I D2 1.35E-41 252.72
  • EIPEEIK (SEQ. ID. No. 128) 856.4542 Bio-I D2 0.026324 81.297
  • EIPEEIKDR SEQ. ID. No. 129
  • 1127.5823 Bio-I D2 4.64E-06 163.15
  • EWNVSYGTALVADR (SEQ. ID. No. 131 ) 1579.7631 Bio-I D2 1.19E-88 310.34
  • MLYLGEEVK (SEQ. ID. No. 143) 1080.5525 Bio-I D2 6.61E-07 164.72
  • RISENLK (SEQ. ID. No. 144) 858.49232 Bio-I D2 0.0084783 130.22
  • VSGVLCELSK (SEQ. ID. No. 147) 1033.5478 Bio-I D2 2.17E-10 176.33
  • VSGVLCELSKDK (SEQ. ID. No. 148) 1276.6697 Bio-I D2 5.90E-07 131.42
  • WPNDVYFQEK (SEQ. ID. No. 150) 1324.6088 Bio-I D2 4.79E-31 227.27
  • the promoter of ST AT 1 was targeted using 4 gRNA sequences as described in Materials and Methods.
  • the forward and reverse oligos for each of these were annealed and cloned into the Bpil site of CRISPR-BiolD producing 4 different CRISPR-BiolD-pSTAT1 vectors. These were co-transfected in HEK293 cells.
  • the cells were provided with biotin and labelled for various periods of time and processed as described in Materials and Methods to identify and quantify biotinylated proteins.
  • a parallel control experiment was conducted using empty CRISPR- BiolD2 vector (one without ST AT1 -specific gRNA).
  • Table 2 presents results from two independent case-control experiments. Several transcription factors, STAT1 itself among them, were detected in as biotinylated proteins in the experiments where STAT1 -specific gRNA was used but not in the control experiment. Table 2. Representative results from 2 CRISPR-BiolD experiments targeting the STAT1 promoter. Label-free quantitation (LFQ) was used to identify proteins detected in the case experiments but not in the control experiments.
  • LFQ Label-free quantitation
  • Control-1 Control-2 statl-1 statl-2 dCas9- 1.34E+09 1.45E+09 1.64E+09 1.70E+09 3191
  • CRISPR-BiolD can enable detection of transcription factors binding to specific genomic loci when sensitive high-resolution mass spectrometry is used to analyse the biotinylated proteins. This is illustrated with analysis of the STAT1 promoter. The sensitivity is enhanced because multiple dCas9-BiolD2 molecules are targeted to the STAT1 promoter (4 different tiled gRNAs were used).
  • the implementation uses a BiolD2 tag fused C-terminal to dCas9 which might also increase the ability of the fusion protein to biotinylate proteins in its vicinity.
  • the purified complex may be used to develop in vitro diagnostic tools.

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Abstract

Des aspects de la présente invention concernent un procédé et des systèmes qui fournissent une capacité étendue, par comparaison avec les techniques existantes, pour étudier les interactions génome-protéome importantes et le repliement et la dynamique de la chromatine dans des eucaryotes supérieurs. En particulier, mais non exclusivement, la présente invention concerne un système impliquant la technologie d'édition de gène CRISPR/Cas en combinaison avec une ou plusieurs techniques d'identification de protéine sur la base de la biotinylation de proximité. Certains modes de réalisation impliquent l'utilisation d'une spectrométrie de masse quantitative et/ou d'un séquençage de nouvelle génération.
PCT/GB2020/052596 2019-11-04 2020-10-15 Identification médiée par crispr de protéines biotinylées et de régions de chromatine Ceased WO2021089984A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210340592A1 (en) * 2020-04-30 2021-11-04 City University Of Hong Kong Method for Determining Long Non-Coding Ribonucleic Acid Interaction Proteins
CN114149980A (zh) * 2021-11-04 2022-03-08 中山大学 一种新型蛋白质生物素连接酶及基于其的邻近标记系统PhastID
CN115050417A (zh) * 2022-06-27 2022-09-13 华南农业大学 一种基于染色质随机折叠过程预测染色质域结构tad的方法
WO2024064818A3 (fr) * 2022-09-21 2025-04-17 The Broad Institute, Inc. Système enzymatique pour le ciblage précis de cellules

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8895307B2 (en) 2007-12-20 2014-11-25 Butamax Advanced Biofuels Llc Ketol-acid reductoisomerase using NADH
US8906616B2 (en) 2012-12-12 2014-12-09 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
US8945839B2 (en) 2012-12-12 2015-02-03 The Broad Institute Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8993233B2 (en) 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
WO2018053053A1 (fr) * 2016-09-13 2018-03-22 The Broad Institute, Inc. Biotinylation dépendant du voisinage et ses utilisations

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8895307B2 (en) 2007-12-20 2014-11-25 Butamax Advanced Biofuels Llc Ketol-acid reductoisomerase using NADH
US8906616B2 (en) 2012-12-12 2014-12-09 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
US8945839B2 (en) 2012-12-12 2015-02-03 The Broad Institute Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8993233B2 (en) 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US8999641B2 (en) 2012-12-12 2015-04-07 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
WO2018053053A1 (fr) * 2016-09-13 2018-03-22 The Broad Institute, Inc. Biotinylation dépendant du voisinage et ses utilisations

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
AUSUBEL ET AL.: "Current protocols in molecular biology", 1990, JOHN WILEY AND SONS
CHENG AWWANG HYANG HSHI LKATZ YTHEUNISSEN TWRANGARAJAN SSHIVALILA CSDADON DBJAENISCH R.: "Multiplexed activation of endogenous genes by CRiSPR-on, an RNA-guided transcriptional activator system", CELL RES., 27 August 2013 (2013-08-27)
DAE IN KIM ET AL: "An improved smaller biotin ligase for BioID proximity labeling", MOLECULAR BIOLOGY OF THE CELL, vol. 27, no. 8, 15 April 2016 (2016-04-15), US, pages 1188 - 1196, XP055763337, ISSN: 1059-1524, DOI: 10.1091/mbc.E15-12-0844 *
ELISABETH SCHMIDTMANN ET AL: "Determination of local chromatin composition by CasID", NUCLEUS, vol. 7, no. 5, 2 September 2016 (2016-09-02), pages 476 - 484, XP055552115, ISSN: 1949-1034, DOI: 10.1080/19491034.2016.1239000 *
ESCOBAR THELMA M ET AL: "Active and Repressed Chromatin Domains Exhibit Distinct Nucleosome Segregation during DNA Replication", CELL, ELSEVIER, AMSTERDAM NL, vol. 179, no. 4, 31 October 2019 (2019-10-31), pages 953, XP085886750, ISSN: 0092-8674, [retrieved on 20191031], DOI: 10.1016/J.CELL.2019.10.009 *
JINEK ET AL., SCIENCE, vol. 337, 2012, pages 816 - 821
JOSIPOVIC GORAN ET AL: "Active fusions of Cas9 orthologs", JOURNAL OF BIOTECHNOLOGY, vol. 301, 31 May 2019 (2019-05-31), pages 18 - 23, XP085720597, ISSN: 0168-1656, DOI: 10.1016/J.JBIOTEC.2019.05.306 *
JUO, PEI-SHOW: "the Concise Dictionary of Biomedicine and Molecular Biology", 2002, CRC PRESS
KIM DIJENSEN SCNOBLE KA, KC BROUX KHMOTAMEDCHABOKI KROUX KJ.: "An improved smaller biotin ligase for BioID proximity labeling", MOL BIOL CELL, 24 February 2016 (2016-02-24)
KIM ET AL., MOL. BIOL. CELL, 24 February 2016 (2016-02-24), pages 1188 - 1196
METODIEVA GADOKI SLAUSEN BMETODIEV MV.: "Decreased Usage of Specific Scrib Exons Defines a More Malignant Phenotype of Breast Cancer With Worsened Survival", EBIOMEDICINE, vol. 8, June 2016 (2016-06-01), pages 150 - 158
METODIEVA GNOGUEIRA-DE-SOUZA NCGREENWOOD CAL-JANABI KLENG LBUCALA RMETODIEV MV: "CD74-dependent deregulation of the tumor suppressor scribble in human epithelial and breast cancer cells", NEOPLASIA, vol. 15, no. 6, June 2013 (2013-06-01), pages 660 - 8, XP055714829, DOI: 10.1593/neo.13464
SAMBROOK ET AL.: "Molecular Cloning, A Laboratory manual", 2001, COLD HARBOR-LABORATORY PRESS
TOBIAS ANTON ET AL: "Applications of the CRISPR/Cas system beyond gene editing", BIOLOGY METHODS AND PROTOCOLS, vol. 3, no. 1, 1 January 2018 (2018-01-01), XP055680334, DOI: 10.1093/biomethods/bpy002 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210340592A1 (en) * 2020-04-30 2021-11-04 City University Of Hong Kong Method for Determining Long Non-Coding Ribonucleic Acid Interaction Proteins
CN114149980A (zh) * 2021-11-04 2022-03-08 中山大学 一种新型蛋白质生物素连接酶及基于其的邻近标记系统PhastID
CN114149980B (zh) * 2021-11-04 2023-10-13 中山大学 一种新型蛋白质生物素连接酶及基于其的邻近标记系统PhastID
CN115050417A (zh) * 2022-06-27 2022-09-13 华南农业大学 一种基于染色质随机折叠过程预测染色质域结构tad的方法
WO2024064818A3 (fr) * 2022-09-21 2025-04-17 The Broad Institute, Inc. Système enzymatique pour le ciblage précis de cellules

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