WO2017183724A1 - ゲノム配列改変技術における変異導入効率の向上方法、及びそれに用いる分子複合体 - Google Patents
ゲノム配列改変技術における変異導入効率の向上方法、及びそれに用いる分子複合体 Download PDFInfo
- Publication number
- WO2017183724A1 WO2017183724A1 PCT/JP2017/016105 JP2017016105W WO2017183724A1 WO 2017183724 A1 WO2017183724 A1 WO 2017183724A1 JP 2017016105 W JP2017016105 W JP 2017016105W WO 2017183724 A1 WO2017183724 A1 WO 2017183724A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- dna
- nucleic acid
- double
- stranded dna
- complex
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 108
- 230000035772 mutation Effects 0.000 title description 55
- 230000001965 increasing effect Effects 0.000 title description 7
- 230000004048 modification Effects 0.000 title description 6
- 238000012986 modification Methods 0.000 title description 6
- 108020004414 DNA Proteins 0.000 claims abstract description 166
- 239000002773 nucleotide Substances 0.000 claims abstract description 111
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 111
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 93
- 102000053602 DNA Human genes 0.000 claims abstract description 68
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 44
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 44
- 238000012258 culturing Methods 0.000 claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims description 136
- 102000004190 Enzymes Human genes 0.000 claims description 99
- 108090000790 Enzymes Proteins 0.000 claims description 99
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 65
- 230000033590 base-excision repair Effects 0.000 claims description 65
- 239000003112 inhibitor Substances 0.000 claims description 45
- 230000007018 DNA scission Effects 0.000 claims description 12
- 210000004962 mammalian cell Anatomy 0.000 claims description 10
- 102100026846 Cytidine deaminase Human genes 0.000 claims description 9
- 108010031325 Cytidine deaminase Proteins 0.000 claims description 9
- 230000008439 repair process Effects 0.000 claims description 9
- 108010072685 Uracil-DNA Glycosidase Proteins 0.000 claims description 5
- 238000003776 cleavage reaction Methods 0.000 claims description 3
- 230000007017 scission Effects 0.000 claims description 3
- 102000006943 Uracil-DNA Glycosidase Human genes 0.000 claims 1
- 230000027455 binding Effects 0.000 abstract description 12
- 229940088598 enzyme Drugs 0.000 description 94
- 108090000623 proteins and genes Proteins 0.000 description 58
- 239000002609 medium Substances 0.000 description 30
- 231100000350 mutagenesis Toxicity 0.000 description 28
- 238000002703 mutagenesis Methods 0.000 description 27
- 102000004169 proteins and genes Human genes 0.000 description 25
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 24
- 238000010362 genome editing Methods 0.000 description 24
- 235000018102 proteins Nutrition 0.000 description 24
- 239000013612 plasmid Substances 0.000 description 21
- 108091033409 CRISPR Proteins 0.000 description 20
- 230000014509 gene expression Effects 0.000 description 20
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 16
- 229910052725 zinc Inorganic materials 0.000 description 16
- 239000011701 zinc Substances 0.000 description 16
- 241000238631 Hexapoda Species 0.000 description 15
- 238000005516 engineering process Methods 0.000 description 15
- 239000013604 expression vector Substances 0.000 description 15
- 230000037361 pathway Effects 0.000 description 15
- 239000013598 vector Substances 0.000 description 15
- 241000588724 Escherichia coli Species 0.000 description 14
- 108020005004 Guide RNA Proteins 0.000 description 14
- 230000000295 complement effect Effects 0.000 description 14
- 230000000694 effects Effects 0.000 description 14
- 150000001413 amino acids Chemical class 0.000 description 13
- 230000005782 double-strand break Effects 0.000 description 12
- 108010073062 Transcription Activator-Like Effectors Proteins 0.000 description 11
- 239000012634 fragment Substances 0.000 description 11
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 10
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 10
- 108091028113 Trans-activating crRNA Proteins 0.000 description 10
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 10
- 108020001507 fusion proteins Proteins 0.000 description 10
- 102000037865 fusion proteins Human genes 0.000 description 10
- 230000002401 inhibitory effect Effects 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 9
- 108020004999 messenger RNA Proteins 0.000 description 9
- 108020004705 Codon Proteins 0.000 description 8
- 108020001738 DNA Glycosylase Proteins 0.000 description 8
- 102000028381 DNA glycosylase Human genes 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 8
- 241000282414 Homo sapiens Species 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 108010063362 DNA-(Apurinic or Apyrimidinic Site) Lyase Proteins 0.000 description 7
- 102000010719 DNA-(Apurinic or Apyrimidinic Site) Lyase Human genes 0.000 description 7
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000010586 diagram Methods 0.000 description 7
- 238000006467 substitution reaction Methods 0.000 description 7
- 241000193830 Bacillus <bacterium> Species 0.000 description 6
- 102220605874 Cytosolic arginine sensor for mTORC1 subunit 2_D10A_mutation Human genes 0.000 description 6
- 101710163270 Nuclease Proteins 0.000 description 6
- 101000910035 Streptococcus pyogenes serotype M1 CRISPR-associated endonuclease Cas9/Csn1 Proteins 0.000 description 6
- 210000004102 animal cell Anatomy 0.000 description 6
- 210000004900 c-terminal fragment Anatomy 0.000 description 6
- 210000004898 n-terminal fragment Anatomy 0.000 description 6
- 230000036961 partial effect Effects 0.000 description 6
- WYWHKKSPHMUBEB-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 6
- 101100139907 Arabidopsis thaliana RAR1 gene Proteins 0.000 description 5
- 244000063299 Bacillus subtilis Species 0.000 description 5
- 235000014469 Bacillus subtilis Nutrition 0.000 description 5
- 241000701022 Cytomegalovirus Species 0.000 description 5
- 230000004568 DNA-binding Effects 0.000 description 5
- 101100028790 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PBS2 gene Proteins 0.000 description 5
- 102100037111 Uracil-DNA glycosylase Human genes 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 230000000692 anti-sense effect Effects 0.000 description 5
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 229940104302 cytosine Drugs 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 238000003780 insertion Methods 0.000 description 5
- 230000037431 insertion Effects 0.000 description 5
- 230000017730 intein-mediated protein splicing Effects 0.000 description 5
- 239000013615 primer Substances 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 241000282693 Cercopithecidae Species 0.000 description 4
- 101001048956 Homo sapiens Homeobox protein EMX1 Proteins 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 3
- 101100452003 Caenorhabditis elegans ape-1 gene Proteins 0.000 description 3
- 108700010070 Codon Usage Proteins 0.000 description 3
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 3
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 3
- 102100023823 Homeobox protein EMX1 Human genes 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 101100445099 Mus musculus Emx1 gene Proteins 0.000 description 3
- 241000251745 Petromyzon marinus Species 0.000 description 3
- 241000714474 Rous sarcoma virus Species 0.000 description 3
- 238000012300 Sequence Analysis Methods 0.000 description 3
- 108020004459 Small interfering RNA Proteins 0.000 description 3
- 241000193996 Streptococcus pyogenes Species 0.000 description 3
- 108091023040 Transcription factor Proteins 0.000 description 3
- 102000040945 Transcription factor Human genes 0.000 description 3
- 101710172430 Uracil-DNA glycosylase inhibitor Proteins 0.000 description 3
- 238000005273 aeration Methods 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000001638 lipofection Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 229960003087 tioguanine Drugs 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 241001515965 unidentified phage Species 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 208000035657 Abasia Diseases 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- 241000255789 Bombyx mori Species 0.000 description 2
- 241000409811 Bombyx mori nucleopolyhedrovirus Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 2
- 102000000311 Cytosine Deaminase Human genes 0.000 description 2
- 108010080611 Cytosine Deaminase Proteins 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108020001019 DNA Primers Proteins 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 2
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- 101000860092 Francisella tularensis subsp. novicida (strain U112) CRISPR-associated endonuclease Cas12a Proteins 0.000 description 2
- 102100026406 G/T mismatch-specific thymine DNA glycosylase Human genes 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 241000235789 Hyperoartia Species 0.000 description 2
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000713869 Moloney murine leukemia virus Species 0.000 description 2
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 2
- 102100023712 Poly [ADP-ribose] polymerase 1 Human genes 0.000 description 2
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 2
- 102100022433 Single-stranded DNA cytosine deaminase Human genes 0.000 description 2
- 108091027967 Small hairpin RNA Proteins 0.000 description 2
- 238000010459 TALEN Methods 0.000 description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 2
- 241000255993 Trichoplusia ni Species 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- -1 alkyl guanine Chemical compound 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000006481 deamination reaction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 238000012239 gene modification Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000005937 nuclear translocation Effects 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000004055 small Interfering RNA Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- BFFPVEVGHKMWLT-UHFFFAOYSA-N 2-amino-3,7-dihydropurin-6-one;3,7-dihydropurin-6-one Chemical compound O=C1NC=NC2=C1NC=N2.O=C1NC(N)=NC2=C1NC=N2 BFFPVEVGHKMWLT-UHFFFAOYSA-N 0.000 description 1
- WMHLZRDNWFNTCU-UHFFFAOYSA-N 2-nitroso-3,7-dihydropurin-6-one Chemical compound O=C1NC(N=O)=NC2=C1N=CN2 WMHLZRDNWFNTCU-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- 108010049290 ADP Ribose Transferases Proteins 0.000 description 1
- 108010029988 AICDA (activation-induced cytidine deaminase) Proteins 0.000 description 1
- 241000093740 Acidaminococcus sp. Species 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 102000055025 Adenosine deaminases Human genes 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 101100028789 Arabidopsis thaliana PBS1 gene Proteins 0.000 description 1
- 101100365087 Arabidopsis thaliana SCRA gene Proteins 0.000 description 1
- 101100365680 Arabidopsis thaliana SGT1B gene Proteins 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108091092236 Chimeric RNA Proteins 0.000 description 1
- 108020004998 Chloroplast DNA Proteins 0.000 description 1
- 208000037088 Chromosome Breakage Diseases 0.000 description 1
- 101100417900 Clostridium acetobutylicum (strain ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787) rbr3A gene Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 230000008265 DNA repair mechanism Effects 0.000 description 1
- 102100037377 DNA-(apurinic or apyrimidinic site) endonuclease 2 Human genes 0.000 description 1
- 102100035619 DNA-(apurinic or apyrimidinic site) lyase Human genes 0.000 description 1
- 241000252212 Danio rerio Species 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 240000006497 Dianthus caryophyllus Species 0.000 description 1
- 235000009355 Dianthus caryophyllus Nutrition 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 101100379225 Drosophila melanogaster cass gene Proteins 0.000 description 1
- 208000035859 Drug effect increased Diseases 0.000 description 1
- 102100028778 Endonuclease 8-like 1 Human genes 0.000 description 1
- 102100028779 Endonuclease 8-like 2 Human genes 0.000 description 1
- 102100028773 Endonuclease 8-like 3 Human genes 0.000 description 1
- 102100021710 Endonuclease III-like protein 1 Human genes 0.000 description 1
- 241001452028 Escherichia coli DH1 Species 0.000 description 1
- 241001131785 Escherichia coli HB101 Species 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000511010 Eustoma Species 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 241000588088 Francisella tularensis subsp. novicida U112 Species 0.000 description 1
- 108010001498 Galectin 1 Proteins 0.000 description 1
- 102100021736 Galectin-1 Human genes 0.000 description 1
- 208000034951 Genetic Translocation Diseases 0.000 description 1
- 108010005440 Guanosine deaminase Proteins 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000927847 Homo sapiens DNA ligase 3 Proteins 0.000 description 1
- 101000806823 Homo sapiens DNA-(apurinic or apyrimidinic site) endonuclease 2 Proteins 0.000 description 1
- 101001137256 Homo sapiens DNA-(apurinic or apyrimidinic site) lyase Proteins 0.000 description 1
- 101001123824 Homo sapiens Endonuclease 8-like 1 Proteins 0.000 description 1
- 101001123823 Homo sapiens Endonuclease 8-like 2 Proteins 0.000 description 1
- 101001123819 Homo sapiens Endonuclease 8-like 3 Proteins 0.000 description 1
- 101000970385 Homo sapiens Endonuclease III-like protein 1 Proteins 0.000 description 1
- 101000615492 Homo sapiens Methyl-CpG-binding domain protein 4 Proteins 0.000 description 1
- 101001113440 Homo sapiens Poly [ADP-ribose] polymerase 2 Proteins 0.000 description 1
- 101000664956 Homo sapiens Single-strand selective monofunctional uracil DNA glycosylase Proteins 0.000 description 1
- 101000755690 Homo sapiens Single-stranded DNA cytosine deaminase Proteins 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 241000689670 Lachnospiraceae bacterium ND2006 Species 0.000 description 1
- 102100033284 Leucine-rich repeats and immunoglobulin-like domains protein 3 Human genes 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 241000555303 Mamestra brassicae Species 0.000 description 1
- 108091027974 Mature messenger RNA Proteins 0.000 description 1
- 102100021290 Methyl-CpG-binding domain protein 4 Human genes 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 210000004460 N cell Anatomy 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 229930182474 N-glycoside Natural products 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 241000238814 Orthoptera Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 101150034686 PDC gene Proteins 0.000 description 1
- 102000000470 PDZ domains Human genes 0.000 description 1
- 108050008994 PDZ domains Proteins 0.000 description 1
- 101150012394 PHO5 gene Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 108010064218 Poly (ADP-Ribose) Polymerase-1 Proteins 0.000 description 1
- 102100023652 Poly [ADP-ribose] polymerase 2 Human genes 0.000 description 1
- 101710182846 Polyhedrin Proteins 0.000 description 1
- 208000020584 Polyploidy Diseases 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 108091008103 RNA aptamers Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 101150105073 SCR1 gene Proteins 0.000 description 1
- 102000000395 SH3 domains Human genes 0.000 description 1
- 108050008861 SH3 domains Proteins 0.000 description 1
- 229910003797 SPO1 Inorganic materials 0.000 description 1
- 229910003798 SPO2 Inorganic materials 0.000 description 1
- 101100134054 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) NTG1 gene Proteins 0.000 description 1
- 101100150136 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) SPO1 gene Proteins 0.000 description 1
- 101100478210 Schizosaccharomyces pombe (strain 972 / ATCC 24843) spo2 gene Proteins 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 102100038661 Single-strand selective monofunctional uracil DNA glycosylase Human genes 0.000 description 1
- 101710143275 Single-stranded DNA cytosine deaminase Proteins 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 241000256251 Spodoptera frugiperda Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000194020 Streptococcus thermophilus Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 102220548254 Tafazzin_E96Q_mutation Human genes 0.000 description 1
- 208000035199 Tetraploidy Diseases 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 108010035344 Thymine DNA Glycosylase Proteins 0.000 description 1
- 208000026487 Triploidy Diseases 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 102000002258 X-ray Repair Cross Complementing Protein 1 Human genes 0.000 description 1
- 108010000443 X-ray Repair Cross Complementing Protein 1 Proteins 0.000 description 1
- 241000589634 Xanthomonas Species 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 230000037442 genomic alteration Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 239000006451 grace's insect medium Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 239000006870 ms-medium Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 238000010397 one-hybrid screening Methods 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 101150019841 penP gene Proteins 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 101150079601 recA gene Proteins 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000003007 single stranded DNA break Effects 0.000 description 1
- 230000005783 single-strand break Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 101150068906 snr-6 gene Proteins 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 230000000476 thermogenic effect Effects 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
-
- 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 RNAses, DNAses
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
-
- 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
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- 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)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
-
- 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 [CRISPRs]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
- C12Y305/04005—Cytidine deaminase (3.5.4.5)
Definitions
- the present invention is a genome sequence that allows nucleobase modification in a specific region of the genome without double strand breaks in DNA (with no or single strand breaks) and without inserting foreign DNA fragments.
- the present invention relates to a method for improving the efficiency of mutagenesis of a modification technique, and a complex of a nucleic acid sequence recognition module, a nucleobase converting enzyme, and an inhibitor of base excision repair used for the method.
- Non-patent Document 1 a method using an artificial nuclease in which a molecule having a sequence-independent DNA cutting ability and a molecule having a sequence recognition ability are combined has been proposed.
- Patent Document 1 For example, using zinc finger nuclease (ZFN) in which a zinc finger DNA binding domain and a non-specific DNA cleavage domain are linked, a host plant cell or insect cell is recombined at a target locus in DNA ( Patent Document 1), a transcriptional activator-like (TAL) effector that is a DNA binding module of the plant pathogen Xanthomonas genus and a DNA endonuclease linked to TALEN, at a site within or adjacent to a specific nucleotide sequence , A method of cleaving and modifying target genes (Patent Document 2), or the DNA sequence CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) that functions in the acquired immune system of eubacteria and archaea, and plays an important role together with CRISPR CRISPR-Cas9 system combined with nuclease Cas (CRISPR-associated) protein family And a method of use (Patent Document 3)
- an artificial nuclease in which a PPR protein composed of 35 amino acids and a nuclease linked to a PPR protein configured to recognize a specific nucleotide sequence by a sequence of PPR motifs that recognize one nucleobase is used.
- a method for cleaving a target gene in the vicinity of a specific sequence (Patent Document 4) has also been reported.
- these genome editing technologies are basically based on double-stranded DNA breaks (DSB), but they involve unexpected genomic alterations, resulting in strong cytotoxicity and chromosomal translocation. There are common problems such as side effects, loss of reliability in gene therapy, extremely few surviving cells due to nucleotide modification, and difficulty in genetic modification itself with primate egg cells or single-cell microorganisms.
- the present inventors used a deaminase that catalyzes a deamination reaction in a CRISPR-Cas system in which at least one DNA cleavage ability of Cas was inactivated. It has been reported that the genome sequence was successfully modified by nucleobase conversion in a region containing a specific DNA sequence without linking DSB by linking the DNA with a molecule capable of recognizing DNA sequence (Patent Document 5). ). According to this genome editing technology, there is no extraneous DNA insertion or DNA double-strand break, so it is safe and the range of mutagenesis is theoretically changed from a single base pinpoint to several hundred bases. It is possible to set a wide range up to a wide range. However, there is a problem that the efficiency of mutagenesis is low as compared with the genome editing technique using Cas9 having normal DNA cutting ability.
- An object of the present invention is to modify a nucleobase of a specific sequence of a gene by cutting a double-stranded DNA without cleavage or by single-strand breakage, a method for genome editing with improved mutagenesis efficiency, and a nucleic acid sequence recognition module therefor It is to provide a complex of a nucleobase converting enzyme and an inhibitor of base excision repair.
- the present inventors sought to develop a method for improving mutation introduction efficiency in a genome editing technique using a nucleobase converting enzyme.
- a method for improving mutagenesis efficiency in general, a method for increasing the nucleobase conversion ability by adding a mutation to a nucleobase converting enzyme or substituting with another enzyme, or a nucleic acid sequence recognition module
- the main focus is on methods for enhancing nucleic acid recognition ability.
- the present inventors changed these general ideas, and in the genome editing technology, one of the causes of low mutagenesis efficiency is base excision repair by DNA glycosylase or the like at the site where the base was converted by the nucleobase converting enzyme.
- PmCDA1 which is a kind of nucleobase converting enzyme
- lamprey which is a thermogenic animal
- the optimal temperature of PmCDA1 enzyme activity is lower than the optimal temperature of about 37 ° C for normal enzymes. It was estimated that the enzyme activity could be increased by adjusting the culture temperature.
- the enzyme activity of PmCDA1 when the cells into which PmCDA1 was introduced were temporarily cultured at a low temperature, it was found that the efficiency of mutagenesis was improved. As a result of further studies based on these findings, the present inventors have completed the present invention.
- the present invention is as follows.
- a method for modifying a targeted site of double-stranded DNA wherein a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the selected double-stranded DNA is bound to PmCDA1 Introducing a complex into a cell having the double-stranded DNA, and culturing the cell at least temporarily at low temperature, thereby cleaving at least one strand of the double-stranded DNA at the targeted site Without converting or deleting one or more nucleotides of the targeted site into one or more other nucleotides, or inserting one or more nucleotides into the targeted site,
- the nucleic acid sequence recognition module is a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated.
- a method for modifying a targeted site of double-stranded DNA comprising a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in selected double-stranded DNA, a nucleobase converting enzyme, A complex bound with an inhibitor of base excision repair is contacted with the double-stranded DNA, and the targeted DNA is cleaved without cleaving at least one strand of the double-stranded DNA at the targeted site.
- nucleic acid sequence recognition module is a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated.
- the Cas lacks two DNA-cleaving abilities.
- nucleobase converting enzyme is cytidine deaminase.
- cytidine deaminase is PmCDA1.
- the mutation introduction efficiency is dramatically improved as compared with the conventional genome editing technology using a nucleobase converting enzyme.
- FIG. 1 is a schematic diagram of the genome editing plasmid used in the examples.
- FIG. 2 is a schematic diagram showing a method for evaluating mutation introduction efficiency.
- FIG. 3 is a diagram showing the results of analyzing the mutation pattern of the target gene region in the obtained mutant population.
- the present invention converts the target nucleotide sequence and its neighboring nucleotides in the double-stranded DNA into other nucleotides without cutting at least one strand of the double-stranded DNA to be modified.
- a method for improving mutagenesis efficiency in a genome editing technique for modifying the targeted site of a double-stranded DNA In the method, a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the double-stranded DNA and a complex bound to PmCDA1 are introduced into a cell having the double-stranded DNA, and the cell is introduced.
- the targeted site that is, the target nucleotide sequence and its neighboring nucleotides are converted to other nucleotides or deleted, or nucleotides are inserted into the site. Including a process.
- the method comprises a complex comprising a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the double-stranded DNA, a nucleobase converting enzyme, and an inhibitor of base excision repair.
- a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the double-stranded DNA
- a nucleobase converting enzyme that specifically binds to a target nucleotide sequence in the double-stranded DNA
- an inhibitor of base excision repair By contacting with the double-stranded DNA, the target site, that is, the target nucleotide sequence and its neighboring nucleotides are converted to other nucleotides or deleted, or nucleotides are inserted into the sites.
- the method comprises: combining the double-stranded DNA with a complex comprising a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the double-stranded DNA and a nucleobase converting enzyme.
- the targeted site i.e., the target nucleotide sequence and its neighboring nucleotides are converted or deleted to other nucleotides by introducing into the cells having and inhibiting base excision repair of the cells Or a step of inserting a nucleotide into the site.
- “modification” of double-stranded DNA means that a certain nucleotide (eg, dC) on the DNA strand is converted to another nucleotide (eg, dT, dA or dG) or deleted. Or a nucleotide or nucleotide sequence inserted between certain nucleotides on a DNA strand.
- the double-stranded DNA to be modified is not particularly limited, but is preferably genomic DNA.
- targeted site of double-stranded DNA refers to all or part of the “target nucleotide sequence” to which the nucleic acid sequence recognition module specifically recognizes and binds, or in the vicinity of the target nucleotide sequence ( One or both of 5 ′ upstream and 3 ′ downstream), and the range thereof can be appropriately adjusted between 1 base and several hundred bases depending on the purpose.
- the “nucleic acid sequence recognition module” means a molecule or molecular complex having the ability to specifically recognize and bind to a specific nucleotide sequence (ie, a target nucleotide sequence) on a DNA strand.
- the nucleic acid sequence recognition module binds to the target nucleotide sequence, so that the nucleobase converting enzyme and the base excision repair inhibitor linked to the module act specifically on the targeted site of double-stranded DNA. enable.
- nucleobase converting enzyme refers to a target nucleotide without cleaving a DNA strand by catalyzing a reaction for converting a substituent on a purine or pyrimidine ring of a DNA base into another group or atom. Is an enzyme capable of converting to other nucleotides.
- base excision repair is one of DNA repair mechanisms possessed by living organisms, and means a mechanism for repairing base damage by cutting out parts damaged by bases and rejoining them. Removal of the damaged base is performed by DNA glycosylase, an enzyme that hydrolyzes the N-glycoside bond of DNA, and the base-free site (apurinic / apyrimidic (AP) site) resulting from the abasic reaction by the enzyme are processed by enzymes downstream of the base excision repair (BER) pathway, such as AP endonuclease, DNA polymerase, DNA ligase.
- BER base excision repair
- UNG NM_003362
- SMUG1 NM_014311
- MBD4 NM_003925
- TDG NM_003211
- OGG1 NM_002542
- MYH NM_012222
- NTHL1 NM_002528
- MPG NM_002434
- NEIL1 NM_024608
- NEIL2 NM_145043
- NEIL3 NM_018248
- APE1 NM_001641
- APE2 NM_014481
- LIG3 NM_013975
- XRCC1 NM_006297
- ADPRT PARP1
- ADPRTL2 PAR0054
- the term “inhibitor of base excision repair” refers to inhibiting BER by inhibiting any stage of the BER pathway or by inhibiting the expression of molecules mobilized in the BER pathway. Means a protein that Further, in the present invention, “inhibiting base excision repair” means inhibiting any stage of the BER pathway, or inhibiting the expression of molecules mobilized in the BER pathway, resulting in Means inhibiting BER.
- the “nucleic acid modifying enzyme complex” means a nucleobase converting enzyme activity having a specific nucleotide sequence recognizing ability, comprising a complex in which the nucleic acid sequence recognizing module and the nucleobase converting enzyme are linked. Means a molecular complex having The complex may further be linked with an inhibitor of base excision repair.
- the “complex” includes not only those composed of a plurality of molecules but also those having molecules constituting the nucleic acid modifying enzyme complex in a single molecule, such as fusion proteins. .
- “encoding a complex” includes both encoding each molecule constituting the complex and encoding a fusion protein having the constituting molecule in a single molecule.
- low temperature means a temperature lower than a general culture temperature for cell growth in cell culture.
- the general culture temperature of cells is 37 ° C.
- the temperature is lower than 37 ° C.
- the culture temperature is too low, the cells are damaged, so the low temperature needs to be a temperature that does not damage the cells.
- the low temperature varies depending on the cell type, culture time and other culture conditions.
- the cell is a mammalian cell such as a Chinese hamster ovary (CHO) cell, it is typically 20 ° C. to 35 ° C., preferably 20 ° C. to 30 ° C. ° C, more preferably 20 ° C to 25 ° C, still more preferably 25 ° C.
- “at least temporarily cultured at a low temperature” means culturing cells at least part of the entire culture period under the above-mentioned “low temperature” conditions, and culturing at a low temperature for the entire culture period. This is also included.
- the case where cells are cultured intermittently at a low temperature divided into a plurality of times during the culture period is also included in “at least temporarily cultured at a low temperature”.
- the timing and duration of the low-temperature culture are not particularly limited, but usually, the low-temperature culture is maintained for overnight or longer after the complex of the nucleic acid sequence recognition module and PmCDA1 or the nucleic acid encoding the complex is introduced into the cells.
- the upper limit of the culture period is not particularly limited as long as it is a minimum period required to modify the targeted site of double-stranded DNA, and may be cultured at a low temperature for the entire culture period.
- the total culture period varies depending on the cell type, culture time, and other culture conditions, but is typically about 10 to 14 days when culturing mammalian cells such as CHO cells at 25 ° C.
- mammalian cells such as CHO cells are 20 ° C. to 35 ° C., preferably 20 ° C. to 30 ° C., more preferably 20 ° C. to 25 ° C., more preferably 25 ° C. after introduction of the complex. Incubation is performed at 0 ° C. for one night or more, preferably overnight to 7 days (for example, overnight).
- the nucleobase converting enzyme used in the present invention is not particularly limited as long as it can catalyze the above reaction.
- a nucleic acid / nucleotide deaminase superfamily that catalyzes a deamination reaction for converting an amino group into a carbonyl group.
- Deaminase belonging to Preferred examples include cytidine deaminase that can convert cytosine or 5-methylcytosine to uracil or thymine, adenosine deaminase that can convert adenine to hypoxanthine, and guanosine deaminase that can convert guanine to xanthine.
- cytidine deaminase include activation-induced cytidine deaminase (hereinafter also referred to as AID), which is an enzyme that introduces a mutation into an immunoglobulin gene in acquired immunity of vertebrates.
- AID activation-induced cytidine deaminase
- PmCDA1 is preferable from the viewpoint of enzyme activity. Further, as shown in Examples described later, it has been found that in a specific embodiment using PmCDA1 as cytidine deaminase, the risk of off-target mutation can be suppressed even when Ugi is used in combination. Therefore, PmCDA1 is preferable from the viewpoint of reducing the risk of off-target mutation.
- the base excision repair inhibitor used in the present invention is not particularly limited as long as it inhibits BER as a result, but from the viewpoint of efficiency, an inhibitor of DNA glycosylase located upstream of the BER pathway is preferable.
- inhibitors of DNA glycosylase used in the present invention include inhibitors of thymine DNA glycosylase, inhibitors of uracil DNA glycosylase, inhibitors of oxoguanine DNA glycosylase, inhibitors of alkyl guanine DNA glycosylase, etc. Not limited.
- cytidine deaminase when used as the nucleobase converting enzyme, it is suitable to use an inhibitor of uracil DNA glycosylase in order to inhibit the repair of U: G or G: U mismatch of DNA caused by mutation. .
- uracil DNA glycosylase inhibitor Ugi
- PBS1 which is a Bacillus subtilis bacteriophage
- uracil DNA glycosylase inhibitor derived from PBS2 which is a Bacillus subtilis bacteriophage (Ugi).
- PBS2-derived Ugi is also known to have an effect of making it difficult to cause mutation or cleavage other than C to T on DNA and recombination, so it is suitable to use PBS2-derived Ugi.
- AP endonuclease nicks the abasic site (AP site) and AP site is completely removed by exonuclease. Is done.
- DNA polymerase creates a new base using the opposite strand as a template, and finally DNA ligase fills the nick and completes the repair.
- Mutant AP endonucleases that have lost enzymatic activity but retain the ability to bind to the AP site are known to competitively inhibit BER. Therefore, these mutant AP endonucleases can also be used as inhibitors of base excision repair of the present invention.
- mutant AP endonuclease is not particularly limited.
- an AP endonuclease derived from E. coli, yeast, mammals eg, human, mouse, pig, cow, horse, monkey, etc.
- the amino acid sequence of human Ape1 can be referred to as UniprotKB No. P27695.
- mutant AP endonucleases that have lost enzyme activity but retain the ability to bind to the AP site include proteins in which the active site or cofactor Mg binding site is mutated.
- E96Q, Y171A, Y171F, Y171H, D210N, D210A, N212A and the like can be mentioned.
- Inhibition of cell base excision repair can be carried out by introducing the above-described BER inhibitor or a nucleic acid encoding the same, or by introducing a low-molecular compound that inhibits BER.
- cell BER can be inhibited by suppressing the expression of genes involved in the BER pathway. Suppression of gene expression is performed, for example, by introducing into the cell an expression vector capable of expressing siRNA, antisense nucleic acid, or these polynucleotides that can specifically suppress the expression of genes involved in the BER pathway. Can do.
- gene expression can be suppressed by knocking out a gene involved in the BER pathway.
- a complex in which a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in a target double-stranded DNA and a nucleobase converting enzyme are bound to each other is used as the double-stranded DNA.
- a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in a target double-stranded DNA and a nucleobase converting enzyme are bound to each other is used as the double-stranded DNA.
- SiRNA is typically a double-stranded oligo RNA composed of RNA having a complementary sequence to the nucleotide sequence of mRNA of a target gene or a partial sequence thereof (hereinafter referred to as target nucleotide sequence) and its complementary strand.
- target nucleotide sequence a partial sequence thereof
- the nucleotide sequences of these RNAs can be appropriately designed based on the sequence information of genes involved in the BER pathway.
- An RNA (small hairpin RNA: shRNA) in which the first sequence forms a double-stranded structure with the second sequence by adopting the structure is also a preferred embodiment of siRNA.
- An antisense nucleic acid includes a nucleotide sequence that can specifically hybridize with the target mRNA under physiological conditions of a cell that expresses the target mRNA (mature mRNA or initial transcript), and the target nucleic acid in a hybridized state. It means a nucleic acid capable of inhibiting translation of a polypeptide encoded by mRNA.
- the type of the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera.
- the nucleotide sequences of these nucleic acids can be appropriately designed based on the sequence information of genes involved in the BER pathway.
- the knockout of a gene involved in the BER pathway means that all or part of the gene involved in the BER pathway has been destroyed or recombined so as not to exhibit its original function.
- the gene may be disrupted or mutated so that one allele on the genome does not function, or a plurality of alleles may be disrupted or mutated.
- Knockout can be performed by a known method, for example, a method of knocking out by introducing into a cell a DNA construct prepared so that genetic recombination with a target gene occurs, TALEN or CRISPR-Cas9 Examples of the method include knocking out by insertion, deletion, and substitution of bases using a system or the like.
- the target nucleotide sequence in the double-stranded DNA that is recognized by the nucleic acid sequence recognition module of the nucleic acid modifying enzyme complex of the present invention is not particularly limited as long as the module can specifically bind, and the target nucleotide sequence in the double-stranded DNA is not limited. It can be any sequence.
- the length of the target nucleotide sequence only needs to be sufficient for the nucleic acid sequence recognition module to specifically bind. For example, when a mutation is introduced at a specific site in mammalian genomic DNA, it depends on the genome size. 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 17 nucleotides or more.
- the upper limit of the length is not particularly limited, but is preferably 25 nucleotides or less, more preferably 22 nucleotides or less.
- the nucleic acid sequence recognition module of the nucleic acid modifying enzyme complex of the present invention includes, for example, a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated (hereinafter also referred to as “CRISPR-mutant Cas”, CRISPR-mutant Cpf1).
- CRISPR-mutant Cas CRISPR-mutant Cpf1
- zinc finger motifs, TAL effector and PPR motifs, etc. including DNA binding domains of proteins that can specifically bind to restriction enzymes, transcription factors, RNA polymerases, etc.
- DNA double-strand breaks Fragments that do not have the ability can be used, but are not limited thereto.
- CRISPR-mutated Cas, zinc finger motif, TAL effector, PPR motif and the like can be mentioned.
- CRISPR-Cas9 In addition to CRISPR-Cas9, an example using CRISPR-Cpf1 has been reported as a genome editing technique using CRISPR (Zetsche.B., Et al., Cell, 163: 759-771 (2015)).
- Cpf1 is different from Cas9 in that it does not require tracrRNA, cleaved DNA becomes a protruding end, PAM sequence is located on the 5 'side, and is a T-rich sequence.
- Examples of Cpf1 capable of genome editing in mammalian cells include, but are not limited to, Cpf1 derived from Acidaminococcus sp. BV3L6 and Cpf1 derived from Lachnospiraceae bacterium ND2006.
- the mutant Cpf1 lacking the ability to cleave DNA is a D917A mutant in which the 917th Asp residue of Cpf1 (FnCpf1) derived from Francisella novicida U112 is converted with an Ala residue, and the 1006th Glu residue is an Ala residue.
- Converted E1006A mutant, D1255A mutant in which the 1255th Asp residue is converted with an Ala residue, etc. are mentioned, but if it is a mutant Cpf1 lacking DNA cleavage ability, it is not limited to these mutants, It can be used in the present invention.
- the zinc finger motif is a linkage of 3 to 6 different zinc finger units of the Cys2His2 type (one finger recognizes about 3 bases), and can recognize a target nucleotide sequence of 9 to 18 bases.
- Zinc finger motifs are: Modular assembly method (Nat Biotechnol (2002) 20: 135-141), OPEN method (Mol Cell (2008) 31: 294-301), CoDA method (Nat Methods (2011) 8: 67-69) In addition, it can be prepared by a known technique such as E. coli one-hybrid method (Nat Biotechnol (2008) 26: 695-701). The details of the production of the zinc finger motif can be referred to Patent Document 1 described above.
- the TAL effector has a repeating structure of modules of about 34 amino acids, and the binding stability and base specificity are determined by the 12th and 13th amino acid residues (called RVD) of one module.
- RVD 12th and 13th amino acid residues
- the PPR motif consists of 35 amino acids, and is constructed to recognize a specific nucleotide sequence by a series of PPR motifs that recognize one nucleobase.
- the 1st, 4th, and ii (-2) th amino acids of each motif Only recognize the target base. Since there is no dependence on the motif structure and there is no interference from the motifs on both sides, it is possible to produce a PPR protein specific to the target nucleotide sequence by linking the PPR motifs just like the TAL effector.
- Patent Document 4 The details of the production of the PPR motif can be referred to Patent Document 4 above.
- DNA-binding domain of these proteins is well known, so it is easy to design a fragment that contains this domain and does not have the ability to cleave DNA double strands. And can be built.
- nucleic acid sequence recognition modules can be provided as a fusion protein with the above-described nucleobase converting enzyme and / or an inhibitor of base excision repair, or can be a SH3 domain, PDZ domain, GK domain, GB domain, etc.
- Protein binding domains and their binding partners are fused to a nucleic acid sequence recognition module and a nucleobase converting enzyme and / or an inhibitor of base excision repair, respectively, and the protein complex is formed through the interaction between the domain and its binding partner. It may be provided as a body.
- intein can be fused to a nucleic acid sequence recognition module and a nucleobase converting enzyme and / or an inhibitor of base excision repair, and both can be linked by ligation after protein synthesis.
- the contact between the nucleic acid modifying enzyme complex of the present invention and the double-stranded DNA is achieved by introducing the complex or a nucleic acid encoding the complex into a cell having the target double-stranded DNA (eg, genomic DNA).
- a nucleic acid-modifying enzyme complex it is desirable to introduce the nucleic acid-modifying enzyme complex into a cell in the form of a nucleic acid encoding the complex and to express the complex in the cell rather than the nucleic acid-modifying enzyme complex itself. Therefore, a nucleic acid sequence recognition module, a nucleobase converting enzyme, and an inhibitor of base excision repair are translated into a protein as a nucleic acid encoding their fusion protein or using a binding domain or intein, etc.
- nucleic acids encoding them in such a form that a complex can be formed therein.
- the nucleic acid may be DNA or RNA.
- DNA it is preferably double-stranded DNA and is provided in the form of an expression vector placed under the control of a promoter functional in the host cell.
- RNA it is preferably a single-stranded RNA.
- the complex of the present invention does not involve DNA double strand break (DSB), genome editing with low toxicity is possible, and the gene modification method of the present invention can be applied to a wide range of biological materials.
- the cells into which the nucleic acid encoding the nucleic acid-modifying enzyme complex is introduced include mammals such as humans from microbial cells such as bacteria such as Escherichia coli as a prokaryote and yeast as a lower eukaryote. It can encompass cells of any species, including vertebrates, insects, plants, and higher eukaryotic cells.
- a DNA encoding a nucleic acid sequence recognition module such as a zinc finger motif, a TAL effector, and a PPR motif can be obtained by any of the methods described above for each module.
- DNA encoding sequence recognition modules such as restriction enzymes, transcription factors, RNA polymerase, etc. covers the region encoding the desired part of the protein (part containing the DNA binding domain) based on the cDNA sequence information.
- oligo DNA primers can be synthesized and cloned using the total RNA or mRNA fraction prepared from cells producing the protein as a template and amplified by RT-PCR.
- total RNA or mRNA prepared from cells that produce the enzyme by synthesizing oligo DNA primers based on the cDNA sequence information of the enzyme used Cloning can be performed by using the fraction as a template and amplifying by RT-PCR.
- the DNA encoding Ugi derived from PBS2 is designed based on the DNA sequence (accession No. J04434) registered in the NCBI / GenBank database, by designing appropriate primers upstream and downstream of CDS. It can be cloned from the derived mRNA by RT-PCR.
- the cloned DNA can be digested as is or with a restriction enzyme if desired, or a suitable linker (eg, GS linker, GGGAR linker, etc.), spacer (eg, FLAG sequence, etc.) and / or nuclear localization signal (NLS) (If the target double-stranded DNA is mitochondrial or chloroplast DNA, each organelle transfer signal) can be added to prepare DNA encoding the protein.
- a suitable linker eg, GS linker, GGGAR linker, etc.
- spacer eg, FLAG sequence, etc.
- NLS nuclear localization signal
- DNA encoding a fusion protein can be prepared by ligation with DNA encoding a nucleic acid sequence recognition module.
- a nucleic acid-modifying enzyme complex is prepared by fusing a DNA encoding a nucleic acid-modifying enzyme complex with a DNA encoding a binding domain or a binding partner thereof, or by fusing each DNA with a DNA encoding a separated intein. May be allowed to form a complex after being translated in the host cell.
- a linker and / or a nuclear translocation signal can be linked to an appropriate position of each DNA as desired.
- the DNA encoding the nucleic acid-modifying enzyme complex can be synthesized by chemically synthesizing a DNA strand, or by connecting partially synthesized oligo DNA short strands using the PCR method or Gibson-Assembly method. It is also possible to construct DNA encoding the full length.
- the advantage of constructing full-length DNA by chemical synthesis or in combination with PCR method or Gibson Assembly method is that the codon used can be designed over the entire CDS according to the host into which the DNA is introduced. In the expression of heterologous DNA, an increase in protein expression level can be expected by converting the DNA sequence into a codon frequently used in the host organism.
- Data on the frequency of codon usage in the host to be used is, for example, the genetic code usage frequency database (http://www.kazusa.or.jp/codon/index.html) published on the Kazusa DNA Research Institute website. ) May be used, or references describing the frequency of codon usage in each host may be consulted.
- the obtained data and the DNA sequence to be introduced and converting the codon used in the DNA sequence that is not frequently used in the host into a codon that encodes the same amino acid and is frequently used Good.
- An expression vector containing a DNA encoding a nucleic acid modifying enzyme complex can be produced, for example, by ligating the DNA downstream of a promoter in an appropriate expression vector.
- Expression vectors include plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194); yeast-derived plasmids (eg, pSH19, pSH15); insect cell expression Plasmid (eg, pFast-Bac); animal cell expression plasmid (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophage such as ⁇ phage; insect virus vector such as baculovirus ( Examples: BmNPV, AcNPV); animal virus vectors such as retro
- the promoter may be any promoter as long as it is appropriate for the host used for gene expression. In conventional methods involving DSB, the viability of host cells may be significantly reduced due to toxicity, so it is desirable to use an inducible promoter to increase the number of cells by the start of induction. Since sufficient cell growth can be obtained even if the enzyme complex is expressed, a constitutive promoter can also be used without limitation.
- SR ⁇ promoter when the host is an animal cell, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex) Virus thymidine kinase) promoter and the like are used.
- CMV promoter, SR ⁇ promoter and the like are preferable.
- the host is E. coli, trp promoter, lac promoter, recA promoter, .lambda.P L promoter, lpp promoter, T7 promoter and the like are preferable.
- SPO1 promoter When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable.
- yeast When the host is yeast, the Gal1 / 10 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable.
- the host When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
- CaMV35S promoter When the host is a plant cell, CaMV35S promoter, CaMV19S promoter, NOS promoter and the like are preferable.
- an expression vector containing an enhancer, a splicing signal, a terminator, a poly A addition signal, a drug resistance gene, an auxotrophic complementary gene or other selection marker, an origin of replication, etc. can do.
- RNA encoding the nucleic acid-modifying enzyme complex can be prepared by, for example, transcribing to mRNA with a known in vitro transcription system using a vector containing DNA encoding each protein as a template.
- the complex of the present invention can be expressed in the cell.
- the host for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
- the genus Escherichia include, for example, Escherichia coli K12 / DH1 [Proc. Natl. Acad. Sci.
- Bacillus bacteria include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], Bacillus subtilis 207-21 [Journal of Biochemistry, 95, 87 (1984)] and the like.
- yeast examples include Saccharomyces cerevisiae (Saccharomyces cerevisiae) AH22, AH22R - , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe (Schizosaccharomyces pombe) NCYC1913, NCYC2036, Pichia pastoris (Pichia pastoris) KM71 etc. are used.
- Insect cells include, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five TM cells derived from eggs of Trichoplusia ni , Cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used.
- Sf cells include Sf9 cells (ATCC CRL1711), Sf21 cells [above, In Vivo, 13, 213-217 (1977)].
- insects include silkworm larvae, Drosophila and crickets [Nature, 315, 592 (1985)].
- animal cells examples include monkey COS-7 cells, monkey Vero cells, CHO cells, dhfr gene-deficient CHO cells, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells, and human fetuses.
- Cell lines such as kidney-derived cells (eg, HEK293 cells), pluripotent stem cells such as human and other mammalian iPS cells and ES cells, and primary cultured cells prepared from various tissues are used.
- zebrafish embryos, Xenopus oocytes, and the like can also be used.
- Plant cells were prepared from various plants (for example, grains such as rice, wheat and corn, commercial crops such as tomato, cucumber and eggplant, garden plants such as carnation and eustoma, experimental plants such as tobacco and Arabidopsis thaliana). Suspension culture cells, callus, protoplasts, leaf sections, root sections and the like are used. Any of the above host cells may be haploid (haploid) or polyploid (eg, diploid, triploid, tetraploid, etc.).
- the expression vector may be introduced by a known method (eg, lysozyme method, competent method, PEG method, CaCl 2 coprecipitation method, electroporation method, microinjection method, particle gun method, lipofection method, Agrobacterium method, etc.).
- E. coli can be transformed according to the method described in, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982).
- Bacillus can be introduced into a vector in accordance with, for example, the method described in Molecular & General Genetics, 168, 111 (1979).
- Yeast can be introduced with a vector according to the method described in, for example, Methods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978). Insect cells and insects can be introduced with a vector according to the method described in, for example, Bio / Technology, 6, 47-55 (1988). Animal cells can be introduced according to the method described in, for example, Cell Engineering Supplement 8 New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973).
- Culture of cells into which a vector has been introduced can be performed according to a known method depending on the type of host.
- a liquid medium is preferable as the medium used for the culture.
- a culture medium contains a carbon source, a nitrogen source, an inorganic substance, etc. which are required for the growth of a transformant.
- the carbon source include glucose, dextrin, soluble starch, and sucrose
- examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, Inorganic or organic substances such as potato extract
- examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like.
- yeast extract, vitamins, growth promoting factors and the like may be added to the medium.
- the pH of the medium is preferably about 5 to about 8.
- M9 medium containing glucose and casamino acids As a medium for culturing Escherichia coli, for example, M9 medium containing glucose and casamino acids [Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] is preferable. If necessary, an agent such as 3 ⁇ -indolylacrylic acid may be added to the medium in order to make the promoter work efficiently.
- Cultivation of E. coli is usually performed at about 15 to about 43 ° C. If necessary, aeration or agitation may be performed.
- the culture of Bacillus is usually performed at about 30 to about 40 ° C. If necessary, aeration or agitation may be performed.
- a medium for culturing yeast for example, a Burkholder minimum medium (Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)) and an SD medium containing 0.5% casamino acid [Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)].
- the pH of the medium is preferably about 5 to about 8.
- the culture is usually performed at about 20 ° C to about 35 ° C. Aeration and agitation may be performed as necessary.
- a medium for culturing insect cells or insects for example, a medium appropriately added with an additive such as 10% bovine serum deactivated in Grace's Insect Medium (Nature, 195, 788 (1962)) is used.
- the pH of the medium is preferably about 6.2 to about 6.4.
- the culture is usually performed at about 27 ° C. You may perform ventilation
- a medium for culturing animal cells for example, a minimum essential medium (MEM) containing about 5 to about 20% fetal calf serum (Science, 122, 501 (1952)), Ham's F12 medium (Ham's F12 medium) Dulbecco's Modified Eagle Medium (DMEM) [Virology, 8, 396 (1959)], RPMI-1640 Medium [The Journal of Medicine, American Medicine, Association, 199, 519 (1967)], 199 Medium (Proceeding of Science, Society, Biologic, Medicine , 73, 1 (1950)].
- the pH of the medium is preferably about 6 to about 8.
- the culture is usually performed at about 30 ° C to about 40 ° C. You may perform ventilation
- MS medium As a medium for culturing plant cells, MS medium, LS medium, B5 medium and the like are used.
- the pH of the medium is preferably about 5 to about 8.
- the culture is usually performed at about 20 ° C to about 30 ° C. You may perform ventilation
- the culture period is not particularly limited as long as it is longer than the minimum necessary period for modifying the targeted site of the double-stranded DNA, and can be appropriately selected depending on the host cell.
- the culture is preferably not performed beyond a period sufficient to modify the targeting site in order to avoid undesired off-target mutations.
- the timing and period of the low-temperature culture in the case of performing the step of culturing at low temperature at least temporarily is as described above.
- the nucleic acid modifying enzyme complex can be expressed in cells.
- RNA encoding a nucleic acid-modifying enzyme complex into a host cell can be performed by a microinjection method, a lipofection method, or the like. RNA can be introduced once or repeatedly several times (for example, 2 to 5 times) at an appropriate interval.
- the nucleic acid sequence recognition module When a complex of a nucleic acid sequence recognition module and a nucleobase converting enzyme is expressed from an expression vector or RNA molecule introduced into a cell, the nucleic acid sequence recognition module is contained in the target double-stranded DNA (eg, genomic DNA).
- the target nucleotide sequence (including all or part of the target nucleotide sequence or the vicinity thereof) is specifically recognized and bound by the action of the nucleobase converting enzyme linked to the nucleic acid sequence recognition module.
- Base conversion occurs in the sense strand or antisense strand (adjustable within the range of several hundred bases), and mismatches occur in double-stranded DNA (for example, cytidine deaminase such as PmCDA1 or AID is used as a nucleobase converting enzyme.
- cytidine deaminase such as PmCDA1 or AID is used as a nucleobase converting enzyme.
- the cytosine on the sense or antisense strand at the targeted site is converted to uracil, resulting in a U: G or G: U mismatch. That).
- This mismatch is not repaired correctly, and the opposite strand base is repaired to pair with the converted strand base (TA or AT in the above example), or replaced with another nucleotide during repair.
- Zinc finger motifs are not efficient in producing zinc fingers that specifically bind to the target nucleotide sequence, and the selection of zinc fingers with high binding specificity is complicated, so many zinc finger motifs that actually function are created. It's not easy. TAL effectors and PPR motifs have a higher degree of freedom in target nucleic acid sequence recognition than zinc finger motifs, but it is necessary to design and construct a huge protein each time depending on the target nucleotide sequence, which is problematic in terms of efficiency. Remains. In contrast, the CRISPR-Cas system recognizes the target double-stranded DNA sequence with a guide RNA complementary to the target nucleotide sequence, so it can synthesize oligo DNA that can specifically hybridize with the target nucleotide sequence.
- CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated is used as the nucleic acid sequence recognition module.
- FIG. 1 shows a schematic diagram of a schematic diagram of the plasmid for genome editing of the present invention using CRISPR-mutated Cas as a nucleic acid sequence recognition module.
- the nucleic acid sequence recognition module of the present invention using CRISPR-mutated Cas is an RNA molecule consisting of a guide RNA (gRNA) complementary to a target nucleotide sequence, and a tracrRNA necessary for recruitment of the mutant Cas protein, and the mutant Cas protein. Provided as a complex.
- gRNA guide RNA
- the Cas protein used in the present invention is not particularly limited as long as it belongs to the CRISPR system, but is preferably Cas9.
- Cas9 include, but are not limited to, Cas9 (SpCas9) derived from Streptococcus pyogenes, Cas9 (StCas9) derived from Streptococcus thermophilus, and the like. SpCas9 is preferable.
- the mutant Cas used in the present invention is one in which the ability to cleave both strands of the double-stranded DNA of Cas protein is inactivated, and one having the nickase activity in which only one strand is cleaved is inactivated. It can be used.
- the 10th Asp residue is converted to an Ala residue
- the D10A mutant lacking the ability to cleave the opposite strand that forms a complementary strand with the guide RNA, or the 840th His residue is The H840A mutant lacking the ability to cleave the guide RNA and the complementary strand converted at the Ala residue, or a double mutant thereof, can be used, but other mutant Cass can be used as well.
- the nucleobase converting enzyme and the inhibitor of base excision repair are provided as a complex with mutant Cas by the same method as that for linking with zinc fingers and the like.
- a nucleobase converting enzyme and / or an inhibitor of base excision repair and a mutant Cas can be bound using RNA aptamer MS2F6, PP7 or the like and RNA scaffold by a binding protein thereof.
- the mutant Cas is recruited to the subsequent tracrRNA, and the DNA cleavage site recognition sequence PAM (protospacer ajay cent motif) (SpCas9 is used, PAM is NGG (N is N Nucleobase converting enzyme that recognizes any base) 3 bases and can theoretically target anywhere in the genome) but cannot cleave one or both DNAs and is linked to mutant Cas
- PAM protospacer ajay cent motif
- NGG N is N Nucleobase converting enzyme that recognizes any base 3 bases and can theoretically target anywhere in the genome
- mismatches occur in the double-stranded DNA.
- Various mutations are introduced due to errors in the BER system of cells trying to repair this.
- CRISPR-mutated Cas when used as a nucleic acid sequence recognition module, it has the target double-stranded DNA in the form of a nucleic acid encoding a nucleic acid modifying enzyme complex, as in the case of using zinc fingers or the like as a nucleic acid sequence recognition module. It is desirable to introduce into cells.
- DNA encoding Cas can be cloned from cells producing the enzyme by methods similar to those described above for DNA encoding inhibitors of base excision repair.
- the mutant Cas is obtained by using the site-directed mutagenesis method known per se to the cloned Cas-encoding DNA, and the amino acid residue at the site important for DNA cleavage activity (for example, in the case of Cas9, the 10th position). Asp residues and the 840th His residue, but not limited thereto) can be obtained by introducing mutations so that they are converted with other amino acids.
- the DNA encoding mutant Cas is a host used in combination with chemical synthesis or PCR method or Gibson Assembly method in the same manner as described above for DNA encoding nucleic acid sequence recognition module or DNA glycosylase. It can also be constructed as DNA with codon usage suitable for expression in cells.
- CDS sequence and amino acid sequence obtained by optimizing SpCas9 for expression in eukaryotic cells are shown in SEQ ID NO: 3 and SEQ ID NO: 4.
- SEQ ID NO: 3 the CDS sequence and amino acid sequence obtained by optimizing SpCas9 for expression in eukaryotic cells.
- the DNA encoding the mutant Cas and the DNA encoding the nucleobase converting enzyme may be linked so as to be expressed as a fusion protein, or expressed separately using a binding domain, intein, etc. It may be designed to form a complex in the host cell via protein ligation. Alternatively, the DNA encoding the mutant Cas and the DNA encoding the nucleobase converting enzyme are divided into two fragments at appropriate locations, and one of these fragments is directly or appropriately linked.
- a nucleic acid-modifying enzyme complex is expressed as two partial complexes, and they are assembled and refolded in the cell, whereby a functional mutation having a specific nucleic acid sequence recognition ability
- a functional nucleobase converting enzyme having a nucleobase conversion reaction catalytic activity may be reconstituted.
- DNA encoding the N-terminal fragment of mutant Cas and DNA encoding the C-terminal fragment are prepared by PCR using appropriate primers, respectively, while the N-terminal fragment of nucleobase converting enzyme is prepared.
- DNA encoding and the DNA encoding the C-terminal fragment in the same manner.
- the DNA encoding the N-terminal fragment and the DNA encoding the C-terminal fragment are ligated using conventional methods.
- DNA encoding two partial complexes can be produced.
- the DNA encoding the N-terminal fragment of the mutant Cas and the DNA encoding the C-terminal fragment of the nucleobase converting enzyme, and the DNA encoding the N-terminal fragment of the nucleobase converting enzyme and the mutant Cas It is also possible to prepare DNA encoding two partial complexes by ligating with DNA encoding the C-terminal fragment of.
- Each partial complex may be linked to be expressed as a fusion protein, or may be expressed separately using a binding domain, intein, etc., and the complex may be formed in the host cell via protein-protein interaction or protein ligation. It may be designed to form.
- two partial complexes may be linked so as to be expressed as one fusion protein.
- There are no particular restrictions on the location of the mutant Cas as long as the two fragments can be reconstituted so that they recognize and bind to the target nucleotide sequence. It may be a fragment, or three or more fragments generated by dividing at two or more sites may be appropriately linked to form two fragments.
- the three-dimensional structures of various Cas9 proteins are known, and those skilled in the art can appropriately select a fragmentation site based on the information.
- the region consisting of the 94th to 718th amino acids from the N-terminus is the domain (REC) involved in the recognition of the target nucleotide sequence and guide RNA
- the region consisting of the 1099th to C-terminal amino acids is PAM Is a domain (PI) involved in the interaction of, so it is within any region within the REC domain or within the PI domain, preferably a region having no structure (eg, between the 204th and 205th amino acids from the N-terminus (204 ..205), between the 535th and 536th amino acids from the N-terminus (535..536), etc.) can be divided into N-terminal and C-terminal fragments (eg, NatNBiotechnol. 33).
- a combination of DNA encoding an inhibitor of base excision repair and DNA encoding mutant Cas and / or DNA encoding nucleobase converting enzyme can also be designed in the same
- the DNA encoding the obtained mutant Cas and / or nucleobase converting enzyme and / or inhibitor of base excision repair can be inserted downstream of the promoter of the same expression vector as described above depending on the host.
- the DNA encoding the guide RNA and the tracrRNA contains a nucleotide sequence complementary to the target nucleotide sequence. For example, when FnCpf1 is recruited as Cas, the DNA is 5 ′ to the complementary nucleotide sequence.
- a crRNA containing AAUU UCUAC UGUU GUAGA U can be used, and the underlined sequence forms a base pair to form a stem-loop structure), or a crRNA coding sequence as necessary
- An oligo DNA sequence ligated with a known tracrRNA coding sequence (for example, gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtggtgcttttt; SEQ ID NO: 9) is designed using a DNA / RNA synthesizer. Can be synthesized.
- DNA encoding guide RNA and tracrRNA can also be inserted into an expression vector similar to the above depending on the host, but as a promoter, a pol III promoter (eg, SNR6, SNR52, SCR1, RPR1, U6) , it is preferable to use a H1 promoter, etc.) and a terminator (e.g., T 6 sequences).
- a promoter e.g., SNR6, SNR52, SCR1, RPR1, U6
- a terminator e.g., T 6 sequences
- RNA encoding mutant Cas and / or nucleobase converting enzyme and / or inhibitor of base excision repair encodes, for example, DNA encoding the above mutant Cas and / or nucleobase converting enzyme and / or inhibitor of base excision repair
- the vector to be prepared can be prepared by transcription into mRNA using a known in vitro transcription system.
- the guide RNA-tracrRNA can be chemically synthesized using a DNA / RNA synthesizer by designing an oligo RNA sequence in which a sequence complementary to a target nucleotide sequence and a known tracrRNA sequence are linked.
- DNA or RNA encoding a mutant Cas and / or nucleobase converting enzyme and / or inhibitor of base excision repair may be transferred to the host cell by the same method as described above. Can be introduced.
- DSBs DNA double-strand breaks
- off-target breaks random chromosome breaks
- mutation introduction efficiency can be increased compared to targeting a single nucleotide sequence.
- the effect is similar to the case where both of the target nucleotide sequences overlap, and even when they are separated by about 600 bp, mutation induction is realized in the same manner. It occurs both when the target nucleotide sequence is in the same direction (the target nucleotide sequence is present on the same strand) and when it is opposite (the target nucleotide sequence is present on each strand of the double-stranded DNA). obtain.
- the genome sequence modification method of the present invention can modify a plurality of DNA regions at completely different positions as targets.
- different target nucleotide sequences may be within one target gene or may be within two or more different target genes. These target genes are on the same chromosome.
- Two or more nucleic acid sequence recognition modules that specifically bind to each other may be used.
- each one of these nucleic acid sequence recognition modules, a nucleobase converting enzyme, and an inhibitor of base excision repair form a nucleic acid modifying enzyme complex.
- the same nucleobase converting enzyme and base excision repair inhibitor can be used.
- a common complex including fusion protein
- Cas protein nucleobase converting enzyme, and base excision repair inhibitor
- guide RNA- As tracrRNA two or more kinds of chimeric RNAs each comprising two or more guide RNAs that form complementary strands with different target nucleotide sequences and tracrRNA can be prepared and used.
- each nucleic acid sequence recognition module that specifically binds to a different target nucleotide is fused with a nucleobase converting enzyme and a base excision repair inhibitor. Can be made.
- an expression vector containing DNA encoding the nucleic acid modifying enzyme complex, or RNA encoding the nucleic acid modifying enzyme complex is used.
- an expression vector capable of autonomous replication in the host cell, but since the plasmid etc. are foreign DNA, after successful mutagenesis, It is preferred that it be removed quickly.
- the plasmid introduced from the host cell is removed using various plasmid removal methods well known in the art. It is desirable.
- an expression vector that does not have autonomous replication ability in the host cell for example, a replication origin that functions in the host cell and / or a protein necessary for replication
- Cells were transfected with 1.5 ⁇ g plasmid and 2 ⁇ L Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Five hours after transfection, the medium was changed to Ham F12 medium containing 0.125 mg / mL G418 (InvivoGen, San Diego, CA, USA) and the cells were incubated for 7 days. Then, it used for calculation of the following mutagenesis efficiency.
- the step of culturing at a low temperature temporarily is the same as above.Transfection is performed 5 hours after the transfection, and the medium is replaced with Ham F12 medium containing 0.125 mg / mL G418 and cultured at 25 ° C. overnight. Subsequently, the cells were cultured at 37 ° C. for 2 days. Then, it used for calculation of the following mutagenesis efficiency.
- HPRT Hydroxanthine-guanine phosophoribosyltransferase
- EDTA trypsin-EDTA
- G418 or G418 + 5 g / mL 6-TG Spread on a dish containing Ham F12 medium containing Tokyo Chemical Industry, Tokyo, Japan. After 7 days, the number of resistant colonies was counted. Mutagenesis efficiency was calculated as the ratio of 6TG resistant colonies to G418 resistant colonies.
- Genomic DNA was extracted from the pellets using NucleoSpin Tissue XS kit (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions.
- a PCR fragment containing the HPRT target site was amplified from genomic DNA using a forward primer (ggctacatagagggatcctgtgtca; SEQ ID NO: 18) and a reverse primer (acagtagctcttcagtctgataaa; SEQ ID NO: 19).
- the PCR product was TA cloned into an E. coli vector and analyzed by the Sanger method.
- DNA was processed and constructed by PCR, restriction enzyme treatment, ligation, Gibson Assembly, or artificial chemical synthesis.
- the plasmid was amplified with E. coli strains XL-10 gold or DH5 ⁇ and introduced into the cells by lipofection.
- FIG. 1 A schematic diagram of the plasmid vector for genome editing used in the Examples is shown in FIG. Based on the pcDNA3.1 vector, a vector for gene transfer by transfection into CHO cells was constructed.
- a nuclear translocation signal (ccc aag aag aag agg aag gtg) with a Cas9 gene ORF derived from Streptococcus pyogenes downstream of the CMV promoter and a codon optimized for eukaryotic expression (SEQ ID NO: 3 (encoding SEQ ID NO: 4)) Linking the addition of SEQ ID NO: 11 (PKKKRKV; encoding SEQ ID NO: 12)), and codon optimized for human cell expression of ORF of deaminase gene (PmCDA1 from lamprey Petromyzon marinus) via linker sequence The ones (SEQ ID NO: 1 (encoding SEQ ID NO: 2)) were connected and expressed as a fusion protein.
- Ugi gene Ugi derived from PBS2 with codon optimized for eukaryotic expression: SEQ ID NO: 5 (encoding SEQ ID NO: 6)).
- a sequence encoding 2A peptide gaa ggc agg gga agc ctt ctg act tgt ggg gat gtg gaa gaa aac cct ggt cca; SEQ ID NO: 13 (EGRGSLLTCGDVEENPGP; SEQ ID NO: 1
- the drug resistance gene (NeoR: G418 resistance gene) was also linked via 4)).
- linker sequence 2 ⁇ GS linker (ggt gga gga ggt tct; SEQ ID NO: 15 (GGGGS; encoding SEQ ID NO: 16) was repeated twice) was used.
- a terminator SV40 poly A signal terminator (SEQ ID NO: 17) was connected.
- mutation Cas9 (nCas9) introduced with a mutation that converts the 10th aspartic acid into alanine (D10A, corresponding DNA sequence mutation a29c), and a mutation that converts the 840th histidine into alanine (H840A, corresponding DNA)
- Mutant Cas9 (dCas9) introduced with the sequence mutation ca2518gc) was used to remove the ability to cleave DNA strands on one or both sides, respectively.
- gRNA was arranged between the H1 promoter (SEQ ID NO: 10) and poly T signal (tttttt) as a chimeric structure with tracrRNA (derived from Streptococcus pyogenes; SEQ ID NO: 9), and incorporated into a plasmid vector for expressing the deaminase gene and the like.
- the gRNA target nucleotide sequence is the 16th to 34th sequence (ccgagatgtcatgaaagaga; SEQ ID NO: 7) (site 1) from the start of exon 3 of the HPRT gene, and the -15th to 3rd from the start of exon 1 of the HPRT gene.
- the complementary strand sequence (ccatgacggaatcggtcggc; SEQ ID NO: 8) (site 2R) was used. It was introduced into cells and expressed in the cells to form a complex of gRNA-tracrRNA and Cas9-PmCDA1 or Cas9-PmCDA1-Ugi.
- Example 1 Evaluation of Mutagenesis Efficiency by Various Genome Editing Plasmids and Conditions Table 1 shows the results of evaluating the mutagenesis efficiency by various genome editing plasmids and conditions.
- the site 1 SEQ ID NO: 7
- the mutation introduction efficiency is 35.9%, and in the plasmid using dCas9 as the mutation Cas9 (dCas-PmCDA1-2A-Neo), the mutation introduction efficiency was 2.08%.
- the mutagenesis efficiency is 91.0%, and dCas9 as the mutation Cas9.
- nCas9 nCas-PmCDA1-2A-Neo
- dCas9 dCas-PmCDA1-2A-Neo
- Genomic DNA was extracted from the obtained mutation introduction colony, the target region of HPRT gene was amplified by PCR, TA cloning was performed, and sequence analysis was then performed. The results are shown in FIG.
- the editing vectors used were Cas9, nCas9 (D10A) -PmCDA1, dCas9-PmCDA1, no inhibitors of base excision repair were expressed, and colonies from cells cultured at 37 ° C were used. It was.
- TGG surrounded by a black square represents a PAM sequence.
- Cas9 without mutations large deletions and insertions centered on the PAM sequence were observed.
- nCas9 (D10A) -PmCDA1 a small deletion of about a dozen bases was observed, and the region was a region containing a deaminated target base.
- dCas9-PmCDA1 a mutation from C to T at 19 to 21 bases upstream of the PAM sequence was observed, and a single-point pinpoint mutation was introduced in 10 of all 14 clones whose sequences were analyzed. It was.
- the vector includes a gRNA target nucleotide sequence, the sequence of the EMX1 gene (represented by SEQ ID NO: 21) described in “Tsai SQ et al., (2015) Nat Biotechnol., 33 (2): 187-197” and its off-target. Except for the candidate sequences 1 to 4 (corresponding to the sequences of Emx 1 off target 1 to Emx 1 off target 4 in Tables 2 and 3, respectively, represented by SEQ ID NOs: 22 to 25), the same as in Example 1 Using.
- the vector was introduced into HEK293T cells by transfection, and cell sorting was not performed.
- nCas9-PmCDA1-UGI when used, the mutation rate is improved in the EMX1 target sequence compared to nCas9-PmCDA1 without UGI, but there is almost no difference in the mutation rate in the off-target candidate sequence. Not observed, off-target mutations were suppressed.
- dCas9-PmCDA1-UGI when used, the mutation rate is improved with the target sequence of EMX1 compared to dCas9-PmCDA1 without UGI, but the mutation rate is almost different with the sequence of off-target candidates. Was not observed, and off-target mutations were suppressed.
- the present invention makes it possible to introduce a site-specific mutation into an arbitrary species safely and with high efficiency without insertion of foreign DNA or DNA double-strand break, and is extremely useful.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
例えば、ジンクフィンガーDNA結合ドメインと非特異的なDNA切断ドメインとを連結した、ジンクフィンガーヌクレアーゼ(ZFN)を用い、宿主の植物細胞又は昆虫細胞にDNA中の標的遺伝子座において組換えを行う方法(特許文献1)、植物病原菌キサントモナス属が有するDNA結合モジュールである転写活性化因子様(TAL)エフェクターと、DNAエンドヌクレアーゼとを連結したTALENを用いて、特定のヌクレオチド配列内又はそれに隣接する部位で、標的遺伝子を切断・修飾する方法(特許文献2)、あるいは、真正細菌や古細菌が持つ獲得免疫システムで機能するDNA配列CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)と、CRISPRとともに重要な働きを持つヌクレアーゼCas(CRISPR-associated)タンパク質ファミリーとを組み合わせたCRISPR-Cas9システムを利用する方法(特許文献3)などが報告されている。さらには、35個のアミノ酸からなり1個の核酸塩基を認識するPPRモチーフの連続によって、特定のヌクレオチド配列を認識するように構成されたPPRタンパク質と、ヌクレアーゼとを連結した人工ヌクレアーゼを用い、該特定配列の近傍で標的遺伝子を切断する方法(特許文献4)も報告されている。
しかし、これらのゲノム編集技術は、基本的にDNA二重鎖切断(double-stranded DNA breaks : DSB)を前提としているが、想定外のゲノム改変を伴うため、強い細胞毒性や染色体の転位などの副作用があり、遺伝子治療における信頼性を損なったり、ヌクレオチド改変による生存細胞数が極めて少なかったり、霊長類卵細胞や単細胞微生物では遺伝子改変自体が困難であるといった共通の課題があった。
本発明者らは、これらの知見に基づいてさらに研究を重ねた結果、本発明を完成するに至った。
[1]二本鎖DNAの標的化された部位を改変する方法であって、選択された二本鎖DNA中の標的ヌクレオチド配列と特異的に結合する核酸配列認識モジュールと、PmCDA1とが結合した複合体を、該二本鎖DNAを有する細胞へ導入し、該細胞を少なくとも一時的に低温で培養することにより、該標的化された部位において該二本鎖DNAの少なくとも一方の鎖を切断することなく、該標的化された部位の1以上のヌクレオチドを他の1以上のヌクレオチドに変換する又は欠失させる、あるいは該標的化された部位に1以上のヌクレオチドを挿入する工程を含み、
該核酸配列認識モジュールが、Casの少なくとも1つのDNA切断能が失活したCRISPR-Casシステムである、方法。
[2]前記Casが2つのDNA切断能を欠失したものである、[1]に記載の方法。
[3]前記細胞が哺乳動物細胞である、[1]又は[2]に記載の方法。
[4]低温が20℃ないし35℃である、[3]に記載の方法。
[5]低温が25℃である、[3]に記載の方法。
[6]二本鎖DNAと複合体との接触が、該二本鎖DNAを有する細胞への、該複合体をコードする核酸の導入により行われる、[1]~[5]のいずれかに記載の方法。
[7]二本鎖DNAの標的化された部位を改変する方法であって、選択された二本鎖DNA中の標的ヌクレオチド配列と特異的に結合する核酸配列認識モジュールと、核酸塩基変換酵素と、塩基除去修復のインヒビターとが結合した複合体を、該二本鎖DNAと接触させ、該標的化された部位において該二本鎖DNAの少なくとも一方の鎖を切断することなく、該標的化された部位の1以上のヌクレオチドを他の1以上のヌクレオチドに変換する又は欠失させる、あるいは該標的化された部位に1以上のヌクレオチドを挿入する工程を含み、
該核酸配列認識モジュールが、Casの少なくとも1つのDNA切断能が失活したCRISPR-Casシステムである、方法。
[8]前記Casが2つのDNA切断能を欠失したものである、[7]に記載の方法。
[9]前記核酸塩基変換酵素がシチジンデアミナーゼである、[7]又は[8]に記載の方法。
[10]前記シチジンデアミナーゼがPmCDA1である、[9]に記載の方法。
[11]塩基除去修復のインヒビターがウラシルDNAグリコシラーゼの阻害剤である、[9]又は[10]に記載の方法。
[12]二本鎖DNAと複合体との接触が、該二本鎖DNAを有する細胞への、該複合体をコードする核酸の導入により行われる、[7]~[11]のいずれかに記載の方法。
[13]前記細胞が哺乳動物細胞である、[12]に記載の方法。
[14]二本鎖DNA中の標的ヌクレオチド配列と特異的に結合する核酸配列認識モジュールと、核酸塩基変換酵素と、塩基除去修復のインヒビターとが結合した複合体であって、該核酸配列認識モジュールは、Casの少なくとも1つのDNA切断能が失活したCRISPR-Casシステムであり、標的化された部位において該二本鎖DNAの少なくとも一方の鎖を切断することなく、該標的化された部位の1以上のヌクレオチドを他の1以上のヌクレオチドに変換し又は欠失させ、あるいは該標的化された部位に1以上のヌクレオチドを挿入する、核酸改変酵素複合体。
[15][14]に記載の核酸改変酵素複合体をコードする核酸。
従って、核酸配列認識モジュールと、核酸塩基変換酵素と、塩基除去修復のインヒビターとは、それらの融合タンパク質をコードする核酸として、あるいは、結合ドメインやインテイン等を利用してタンパク質に翻訳後、宿主細胞内で複合体を形成し得るような形態で、それらをそれぞれコードする核酸として調製することが好ましい。ここで核酸は、DNAであってもRNAであってもよい。DNAの場合は、好ましくは二本鎖DNAであり、宿主細胞内で機能的なプロモーターの制御下に配置した発現ベクターの形態で提供される。RNAの場合は、好ましくは一本鎖RNAである。
核酸塩基変換酵素及び塩基除去修復のインヒビターをコードするDNAも、同様に、使用する酵素のcDNA配列情報をもとにオリゴDNAプライマーを合成し、当該酵素を産生する細胞より調製した全RNAもしくはmRNA画分を鋳型として用い、RT-PCR法によって増幅することにより、クローニングすることができる。例えば、PBS2由来のUgiをコードするDNAは、NCBI/GenBankデータベースに登録されているDNA配列(accession No. J04434)をもとに、CDSの上流及び下流に対して適当なプライマーを設計し、PBS2由来mRNAからRT-PCR法によりクローニングできる。
クローン化されたDNAは、そのまま、又は所望により制限酵素で消化するか、適当なリンカー(例、GSリンカー、GGGARリンカー等)、スペーサー(例、FLAG配列等)及び/又は核移行シグナル(NLS)(目的の二本鎖DNAがミトコンドリアや葉緑体DNAの場合は、各オルガネラ移行シグナル)を付加して、タンパク質をコードするDNAを調製することができる。また、さらに核酸配列認識モジュールをコードするDNAとライゲーションして、融合タンパク質をコードするDNAを調製することができる。
あるいは、核酸改変酵素複合体をコードするDNAに、それぞれ結合ドメインもしくはその結合パートナーをコードするDNAを融合させるか、それぞれのDNAに分離インテインをコードするDNAを融合させることにより、核酸改変酵素複合体が宿主細胞内で翻訳された後に複合体を形成できるようにしてもよい。これらの場合も、所望によりそれぞれのDNAの適当な位置に、リンカー及び/又は核移行シグナルを連結することができる。
発現ベクターとしては、大腸菌由来のプラスミド(例、pBR322,pBR325,pUC12,pUC13);枯草菌由来のプラスミド(例、pUB110,pTP5,pC194);酵母由来プラスミド(例、pSH19,pSH15);昆虫細胞発現プラスミド(例:pFast-Bac);動物細胞発現プラスミド(例:pA1-11、pXT1、pRc/CMV、pRc/RSV、pcDNAI/Neo);λファージなどのバクテリオファージ;バキュロウイルスなどの昆虫ウイルスベクター(例:BmNPV、AcNPV);レトロウイルス、ワクシニアウイルス、アデノウイルスなどの動物ウイルスベクターなどが用いられる。
プロモーターとしては、遺伝子の発現に用いる宿主に対応して適切なプロモーターであればいかなるものでもよい。DSBを伴う従来法では毒性のために宿主細胞の生存率が著しく低下する場合があるので、誘導プロモーターを使用して誘導開始までに細胞数を増やしておくことが望ましいが、本発明の核酸改変酵素複合体を発現させても十分な細胞増殖が得られるので、構成プロモーターも制限なく使用することができる。
例えば、宿主が動物細胞である場合、SRαプロモーター、SV40プロモーター、LTRプロモーター、CMV(サイトメガロウイルス)プロモーター、RSV(ラウス肉腫ウイルス)プロモーター、MoMuLV(モロニーマウス白血病ウイルス)LTR、HSV-TK(単純ヘルペスウイルスチミジンキナーゼ)プロモーターなどが用いられる。なかでも、CMVプロモーター、SRαプロモーターなどが好ましい。
宿主が大腸菌である場合、trpプロモーター、lacプロモーター、recAプロモーター、λPLプロモーター、lppプロモーター、T7プロモーターなどが好ましい。
宿主がバチルス属菌である場合、SPO1プロモーター、SPO2プロモーター、penPプロモーターなどが好ましい。
宿主が酵母である場合、Gal1/10プロモーター、PHO5プロモーター、PGKプロモーター、GAPプロモーター、ADHプロモーターなどが好ましい。
宿主が昆虫細胞である場合、ポリヘドリンプロモーター、P10プロモーターなどが好ましい。
宿主が植物細胞である場合、CaMV35Sプロモーター、CaMV19Sプロモーター、NOSプロモーターなどが好ましい。
宿主としては、例えば、エシェリヒア属菌、バチルス属菌、酵母、昆虫細胞、昆虫、動物細胞などが用いられる。
エシェリヒア属菌としては、例えば、エシェリヒア・コリ(Escherichia coli)K12・DH1〔Proc. Natl. Acad. Sci. USA,60,160 (1968)〕,エシェリヒア・コリJM103〔Nucleic Acids Research,9,309 (1981)〕,エシェリヒア・コリJA221〔Journal of Molecular Biology,120,517 (1978)〕,エシェリヒア・コリHB101〔Journal of Molecular Biology,41,459 (1969)〕,エシェリヒア・コリC600〔Genetics,39,440 (1954)〕などが用いられる。
バチルス属菌としては、例えば、バチルス・サブチルス(Bacillus subtilis)MI114〔Gene,24,255 (1983)〕,バチルス・サブチルス207-21〔Journal of Biochemistry,95,87 (1984)〕などが用いられる。
酵母としては、例えば、サッカロマイセス・セレビシエ(Saccharomyces cerevisiae)AH22,AH22R-,NA87-11A,DKD-5D,20B-12、シゾサッカロマイセス・ポンベ(Schizosaccharomyces pombe)NCYC1913,NCYC2036,ピキア・パストリス(Pichia pastoris)KM71などが用いられる。
昆虫としては、例えば、カイコの幼虫、ショウジョウバエ、コオロギなどが用いられる〔Nature,315,592 (1985)〕。
上記いずれの宿主細胞も、半数体(一倍体)であってもよいし、倍数体(例、二倍体、三倍体、四倍体など)であってもよい。
大腸菌は、例えば、Proc. Natl. Acad. Sci. USA,69,2110 (1972)やGene,17,107 (1982)などに記載の方法に従って形質転換することができる。
バチルス属菌は、例えば、Molecular & General Genetics,168,111 (1979)などに記載の方法に従ってベクターを導入することができる。
酵母は、例えば、Methods in Enzymology,194,182-187 (1991)、Proc. Natl. Acad. Sci. USA,75,1929 (1978)などに記載の方法に従ってベクターを導入することができる。
昆虫細胞及び昆虫は、例えば、Bio/Technology,6,47-55 (1988)などに記載の方法に従ってベクターを導入することができる。
動物細胞は、例えば、細胞工学別冊8 新細胞工学実験プロトコール,263-267 (1995)(秀潤社発行)、Virology,52,456 (1973)に記載の方法に従ってベクターを導入することができる。
バチルス属菌の培養は、通常約30~約40℃で行なわれる。必要により、通気や撹拌を行ってもよい。
以上のようにして、核酸改変酵素複合体を細胞内で発現させることができる。
これに対し、CRISPR-Casシステムは、標的ヌクレオチド配列に対して相補的なガイドRNAにより目的の二本鎖DNAの配列を認識するので、標的ヌクレオチド配列と特異的にハイブリッド形成し得るオリゴDNAを合成するだけで、任意の配列を標的化することができる。
従って、本発明のより好ましい実施態様においては、核酸配列認識モジュールとして、Casの少なくとも1つのDNA切断能が失活したCRISPR-Casシステム(CRISPR-変異Cas)が用いられる。
CRISPR-変異Casを用いた本発明の核酸配列認識モジュールは、標的ヌクレオチド配列と相補的なガイドRNA(gRNA)と、変異Casタンパク質のリクルートに必要なtracrRNAとからなるRNA分子と変異Casタンパク質との複合体として提供される。
CasをコードするDNAは、塩基除去修復のインヒビターをコードするDNAについて上記したのと同様の方法により、該酵素を産生する細胞からクローニングすることができる。また、変異Casは、クローン化されたCasをコードするDNAに、自体公知の部位特異的変異誘発法を用いて、DNA切断活性に重要な部位のアミノ酸残基(例えば、Cas9の場合、10番目のAsp残基や840番目のHis残基が挙げられるが、これらに限定されない)を他のアミノ酸で変換するように変異を導入することにより、取得することができる。
あるいは変異CasをコードするDNAは、核酸配列認識モジュールをコードするDNAやDNAグリコシラーゼをコードするDNAについて上記したのと同様の方法により、化学合成又はPCR法もしくはGibson Assembly法との組み合わせで、用いる宿主細胞での発現に適したコドン使用を有するDNAとして構築することもできる。例えば、SpCas9を真核細胞で発現のために最適化したCDS配列及びアミノ酸配列を配列番号3及び配列番号4に示す。配列番号3に示された配列中塩基番号29の「A」を「C」に変換すれば、D10A変異体をコードするDNAを得ることができ、塩基番号2518-2519の「CA」を「GC」に変換すればH840A変異体をコードするDNAを得ることができる。
一方、ガイドRNA及びtracrRNAをコードするDNAは、標的ヌクレオチド配列に対して相補的なヌクレオチド配列を含む、crRNA配列(例えば、CasとしてFnCpf1をリクルートする場合、該相補的なヌクレオチド配列の5’側に配列番号20;AAUUUCUACUGUUGUAGAU を含むcrRNAを用いることができ、下線部の配列同士が塩基対を形成しステム-ループ構造をとる)のコード配列、あるいは、crRNAコード配列と必要に応じて既知のtracrRNAコード配列(例えば、CasとしてCas9をリクルートする場合のtracrRNAコード配列として、gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtggtgctttt; 配列番号9)とを連結したオリゴDNA配列を設計し、DNA/RNA合成機を用いて、化学的に合成することができる。ガイドRNA及びtracrRNAをコードするDNAも、宿主に応じて、上記と同様の発現ベクターに挿入することができるが、プロモーターとしては、pol III系のプロモーター(例、SNR6、SNR52、SCR1、RPR1、U6、H1プロモーター等)及びターミネーター(例、T6配列)を用いることが好ましい。
ガイドRNA-tracrRNAは、標的ヌクレオチド配列に対して相補的な配列と既知のtracrRNA配列とを連結したオリゴRNA配列を設計し、DNA/RNA合成機を用いて、化学的に合成することができる。
あるいは、変異導入に十分な核酸改変酵素複合体の発現が得られる限り、宿主細胞内での自律複製能を有しない発現ベクター(例えば、宿主細胞で機能する複製起点及び/又は複製に必要なタンパク質をコードする遺伝子を欠くベクター等)や、RNAを用いて、一過的発現により目的の二本鎖DNAに変異を導入することもまた好ましい。
<細胞株・培養・形質転換・発現誘導>
チャイニーズハムスター卵巣由来のCHO-K1接着細胞を用いた。細胞を、10%胎仔ウシ血清(Biosera, Nuaille, France)及び100μg/mL ペニシリン-ストレプトマイシン(Life Technologies, Carlsbad, CA, USA)を添加したハムF12培地(Life Technologies, Carlsbad, CA, USA)中で培養した。細胞を加湿した5% CO2雰囲気下において、37℃でインキュベートした。トランスフェクションのため、24ウェルプレートを用い、ウェルあたり0.5×105細胞となるように細胞を播種し、1日間培養した。メーカーの説明書に従い、1.5μg プラスミドと2μL リポフェクタミン 2000(Life Technologies, Carlsbad, CA, USA)を細胞にトランスフェクトした。トランスフェクションから5時間後、培地を0.125mg/mL G418(InvivoGen, San Diego, CA, USA)を含むハムF12培地に交換し、細胞を7日間インキュベートした。その後、以下の変異導入効率の算定に用いた。
変異導入効率の算定の概要図を図2に示した。HPRT(Hypoxanthine-guanine phosophoribosyltransferase)はプリン代謝酵素の1つであり、HPRTの遺伝子が破壊された細胞は、6-TG(6-thioguanine)に対する耐性を獲得する。HPRT遺伝子の変異導入効率を算定するため、トリプシン-EDTA(Life Technologies, Carlsbad, CA, USA)を用いて細胞をプラスチックから剥し、100~500細胞を、G418又はG418 + 5g/mL 6-TG(Tokyo Chemical Industry, Tokyo, Japan)を含むハムF12培地を入れたディッシュ上に広げた。7日後、耐性コロニーの数を計測した。変異導入効率を、G418耐性コロニーに対する6TG耐性コロニーの割合として算定した。
配列解析のため、G418及び6TG耐性コロニーをトリプシン処理し、遠心分離によりペレット状にした。メーカーの説明書に従い、NucleoSpin Tissue XS kit (Macherey-Nagel, Duren, Germany)を用いてペレットからゲノムDNAを抽出した。HPRTの標的部位を含むPCR断片を、フォワードプライマー(ggctacatagagggatcctgtgtca; 配列番号18)及びリバースプライマー(acagtagctcttcagtctgataaaa; 配列番号19)を用いて、ゲノムDNAから増幅した。PCR産物は、大腸菌(E.coli)ベクターにTAクローニングし、サンガー法により解析した。
DNAは、PCR法、制限酵素処理、ライゲーション、Gibson Assembly法、人工化学合成のいずれかによって、加工・構築した。プラスミドは大腸菌株XL-10 goldないしDH5αで増幅し、リポフェクション法で細胞に導入した。
実施例で用いたゲノム編集用プラスミドベクターの概要図を図1に示した。pcDNA3.1ベクターをベースとして、CHO細胞へのトランスフェクションにより遺伝子導入を行うベクターを構築した。CMVプロモーター下流にStreptococcus pyogenes由来のCas9遺伝子ORFを真核細胞発現用にコドンを最適化されたもの(配列番号3(配列番号4をコードする))に核移行シグナル(ccc aag aag aag agg aag gtg; 配列番号11(PKKKRKV; 配列番号12をコードする))を付加したものを繋ぎ、リンカー配列を介してデアミナーゼ遺伝子(ヤツメウナギPetromyzon marinus由来PmCDA1)のORFをヒト細胞発現用にコドンを最適化されたもの(配列番号1(配列番号2をコードする))を繋いで融合タンパク質として発現させた。また、さらにUgi遺伝子(PBS2由来のUgiを真核細胞発現用にコドンを最適化されたもの:配列番号5(配列番号6をコードする))を融合発現するコンストラクトも作成した。また、2Aペプチドをコードする配列(gaa ggc agg gga agc ctt ctg act tgt ggg gat gtg gaa gaa aac cct ggt cca; 配列番号13(EGRGSLLTCGDVEENPGP; 配列番号1
4をコードする))を介して、薬剤耐性遺伝子(NeoR:G418耐性遺伝子)も繋げた。リンカー配列としては、2xGSリンカー(ggt gga gga ggt tct; 配列番号15(GGGGS; 配列番号16をコードする)の2回繰り返し)を用いた。ターミネーターとしてSV40 poly A シグナルターミネーター(配列番号17)を繋いだ。
Cas9において、第10番目のアスパラギン酸をアラニンに変換する変異(D10A、対応DNA配列変異a29c)を導入した変異Cas9(nCas9)、及びさらに840番目のヒスチジンをアラニンに変換する変異(H840A、対応DNA配列変異ca2518gc)を導入した変異Cas9(dCas9)を、それぞれ片側又は両側のDNA鎖の切断能を除去するために用いた。
gRNAはtracrRNA(Streptococcus pyogenes由来; 配列番号9)とのキメラ構造としてH1プロモーター(配列番号10)とpoly Tシグナル(tttttt)の間に配置し、上記デアミナーゼ遺伝子等を発現させるプラスミドベクターに組み込んだ。gRNA標的塩基配列は、HPRT遺伝子のエクソン3の開始点から16番目~34番目の配列(ccgagatgtcatgaaagaga; 配列番号7)(site 1)、及びHPRT遺伝子のエクソン1の開始点から-15番目~3番目の配列の相補鎖配列(ccatgacggaatcggtcggc; 配列番号8)(site 2R)を用いた。細胞に導入し、細胞内にて発現させ、gRNA-tracrRNAと、Cas9-PmCDA1又はCas9-PmCDA1-Ugiとの複合体を形成させた。
様々なゲノム編集プラスミドと条件による変異導入効率を評価した結果を、表1に示す。なお実施例1において、site 2Rとの記載が無いものについては、すべてgRNA標的塩基配列としてsite 1(配列番号7)を用いた。
得られた変異導入コロニーから、ゲノムDNAを抽出して、HPRT遺伝子の標的領域をPCRで増幅し、TAクローニングをしたのち、配列解析を行った。結果を図3に示す。なお、用いた編集ベクターはCas9、nCas9(D10A)-PmCDA1、dCas9-PmCDA1であり、塩基除去修復のインヒビターは発現させておらず、また、培養温度を37℃で行った細胞からのコロニーを用いた。図中、黒四角で囲ったTGGはPAM配列を表す。
変異を導入していないCas9では、PAM配列の直上を中心とした大きな欠失や挿入が認められた。一方で、nCas9(D10A)-PmCDA1では、十数塩基程度の小規模な欠失が認められ、その領域は脱アミノ化標的塩基を含む領域であった。また、dCas9-PmCDA1では、PAM配列の上流19~21塩基にあるCからTへの変異が認められ、配列を解析した全14クローン中の10クローンにおいて、一塩基のピンポイントな変異が導入されていた。
ヒト胎児腎臓由来細胞であるHEK293T細胞を用いて、変異導入効率を評価した。ベクターは、gRNA標的塩基配列を、“Tsai S.Q. et al., (2015) Nat Biotechnol., 33(2): 187-197”に記載のEMX1遺伝子の配列(配列番号21で表す)及びそのオフターゲット候補の配列1~4(それぞれ、表2、3中のEmx 1 off target1~Emx 1 off target4の配列に対応し、配列番号22~25で表す)としたこと以外は実施例1と同じものを用いた。ベクターを、HEK293T細胞にトランスフェクションにより導入し、細胞の選別は行わず、2日後に全細胞を回収してゲノムDNAを抽出した。なお、細胞の選別及び全細胞回収までの期間以外の培養条件、及びトランスフェクションの条件は、上記CHO-K1細胞の条件と同様にした。次いで、“Nishida K. et al. (2016) Science, 6: 353(6305)”に記載の方法に準じて、各標的を含む領域を表2に示すプライマーを用いてPCRによって増幅し、次世代シークエンサーを用いて、変異導入パターンを解析した。結果を表3に示す。表中、配列下の数字はヌクレオチドの置換割合(%)を示す。
Claims (15)
- 二本鎖DNAの標的化された部位を改変する方法であって、選択された二本鎖DNA中の標的ヌクレオチド配列と特異的に結合する核酸配列認識モジュールと、PmCDA1とが結合した複合体を、該二本鎖DNAを有する細胞へ導入し、該細胞を少なくとも一時的に低温で培養することにより、該標的化された部位において該二本鎖DNAの少なくとも一方の鎖を切断することなく、該標的化された部位の1以上のヌクレオチドを他の1以上のヌクレオチドに変換する又は欠失させる、あるいは該標的化された部位に1以上のヌクレオチドを挿入する工程を含み、
該核酸配列認識モジュールが、Casの少なくとも1つのDNA切断能が失活したCRISPR-Casシステムである、方法。 - 前記Casが2つのDNA切断能を欠失したものである、請求項1に記載の方法。
- 前記細胞が哺乳動物細胞である、請求項1又は2に記載の方法。
- 低温が20℃ないし35℃である、請求項3に記載の方法。
- 低温が25℃である、請求項3に記載の方法。
- 二本鎖DNAと複合体との接触が、該二本鎖DNAを有する細胞への、該複合体をコードする核酸の導入により行われる、請求項1~5のいずれか1項に記載の方法。
- 二本鎖DNAの標的化された部位を改変する方法であって、選択された二本鎖DNA中の標的ヌクレオチド配列と特異的に結合する核酸配列認識モジュールと、核酸塩基変換酵素と、塩基除去修復のインヒビターとが結合した複合体を、該二本鎖DNAと接触させ、該標的化された部位において該二本鎖DNAの少なくとも一方の鎖を切断することなく、該標的化された部位の1以上のヌクレオチドを他の1以上のヌクレオチドに変換する又は欠失させる、あるいは該標的化された部位に1以上のヌクレオチドを挿入する工程を含み、
該核酸配列認識モジュールが、Casの少なくとも1つのDNA切断能が失活したCRISPR-Casシステムである、方法。 - 前記Casが2つのDNA切断能を欠失したものである、請求項7に記載の方法。
- 前記核酸塩基変換酵素がシチジンデアミナーゼである、請求項7又は8に記載の方法。
- 前記シチジンデアミナーゼがPmCDA1である、請求項9に記載の方法。
- 塩基除去修復のインヒビターがウラシルDNAグリコシラーゼの阻害剤である、請求項9又は10に記載の方法。
- 二本鎖DNAと複合体との接触が、該二本鎖DNAを有する細胞への、該複合体をコードする核酸の導入により行われる、請求項7~11のいずれか1項に記載の方法。
- 前記細胞が哺乳動物細胞である、請求項12に記載の方法。
- 二本鎖DNA中の標的ヌクレオチド配列と特異的に結合する核酸配列認識モジュールと、核酸塩基変換酵素と、塩基除去修復のインヒビターとが結合した複合体であって、該核酸配列認識モジュールは、Casの少なくとも1つのDNA切断能が失活したCRISPR-Casシステムであり、標的化された部位において該二本鎖DNAの少なくとも一方の鎖を切断することなく、該標的化された部位の1以上のヌクレオチドを他の1以上のヌクレオチドに変換し又は欠失させ、あるいは該標的化された部位に1以上のヌクレオチドを挿入する、核酸改変酵素複合体。
- 請求項14に記載の核酸改変酵素複合体をコードする核酸。
Priority Applications (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/094,587 US20200377910A1 (en) | 2016-04-21 | 2017-04-21 | Method for increasing mutation introduction efficiency in genome sequence modification technique, and molecular complex to be used therefor |
JP2018513234A JP7001272B2 (ja) | 2016-04-21 | 2017-04-21 | ゲノム配列改変技術における変異導入効率の向上方法、及びそれに用いる分子複合体 |
EP17786061.6A EP3447139B1 (en) | 2016-04-21 | 2017-04-21 | Method for increasing mutation introduction efficiency in genome sequence modification technique, and molecular complex to be used therefor |
BR112018071376-7A BR112018071376A2 (pt) | 2016-04-21 | 2017-04-21 | método para modificar um sítio alvejado de um dna de cadeia dupla, complexo enzimático modificador de ácido nucleico, e, ácido nucleico |
SG11201809242VA SG11201809242VA (en) | 2016-04-21 | 2017-04-21 | Method for increasing mutation introduction efficiency in genome sequence modification technique, and molecular complex to be used therefor |
CA3021281A CA3021281C (en) | 2016-04-21 | 2017-04-21 | Method for increasing mutation introduction efficiency in genome sequence modification technique, and molecular complex to be used therefor |
KR1020187032638A KR102116200B1 (ko) | 2016-04-21 | 2017-04-21 | 게놈 서열 변형 기법의 돌연변이 도입 효율을 증가시키는 방법 및 이에 사용되는 분자 복합체 |
DK17786061.6T DK3447139T3 (da) | 2016-04-21 | 2017-04-21 | Fremgangsmåde til øgning af mutationsindføringseffektivitet i genomsekvensmodificeringsteknik, og molekylært kompleks til anvendelse dertil |
CN201780038364.0A CN109312329B (zh) | 2016-04-21 | 2017-04-21 | 提高基因组序列修饰技术中的突变导入效率的方法、及其使用的分子复合体 |
CN202311476665.5A CN117925730A (zh) | 2016-04-21 | 2017-04-21 | 提高基因组序列修饰技术中的突变导入效率的方法、及其使用的分子复合体 |
ES17786061T ES2919961T3 (es) | 2016-04-21 | 2017-04-21 | Método para aumentar la eficiencia de introducción de mutaciones en una técnica de modificación de secuencias genómicas, y complejo molecular para su uso en la misma |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2016-085631 | 2016-04-21 | ||
JP2016085631 | 2016-04-21 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2017183724A1 true WO2017183724A1 (ja) | 2017-10-26 |
Family
ID=60116870
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2017/016105 WO2017183724A1 (ja) | 2016-04-21 | 2017-04-21 | ゲノム配列改変技術における変異導入効率の向上方法、及びそれに用いる分子複合体 |
Country Status (11)
Country | Link |
---|---|
US (1) | US20200377910A1 (ja) |
EP (1) | EP3447139B1 (ja) |
JP (1) | JP7001272B2 (ja) |
KR (1) | KR102116200B1 (ja) |
CN (2) | CN117925730A (ja) |
BR (1) | BR112018071376A2 (ja) |
CA (1) | CA3021281C (ja) |
DK (1) | DK3447139T3 (ja) |
ES (1) | ES2919961T3 (ja) |
SG (1) | SG11201809242VA (ja) |
WO (1) | WO2017183724A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020241869A1 (ja) * | 2019-05-30 | 2020-12-03 | 国立大学法人東京大学 | 2種の核酸塩基変換酵素が融合されたCasタンパク質を利用したゲノム編集システム |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102061438B1 (ko) | 2015-11-27 | 2019-12-31 | 고쿠리츠다이가쿠호진 고베다이가쿠 | 표적화 dna 서열 중의 핵산 염기가 특이적으로 전환되어 있는 단자엽식물 게놈 서열을 전환시키는 방법, 및 그것에 사용되는 분자 복합체 |
US20210032621A1 (en) * | 2018-01-23 | 2021-02-04 | Institute For Basic Science | Extended single guide rna and use thereof |
CN110607320B (zh) * | 2018-11-23 | 2023-05-12 | 电子科技大学 | 一种植物基因组定向碱基编辑骨架载体及其应用 |
WO2021046155A1 (en) | 2019-09-03 | 2021-03-11 | Voyager Therapeutics, Inc. | Vectorized editing of nucleic acids to correct overt mutations |
KR20220097408A (ko) * | 2019-10-17 | 2022-07-07 | 페어와이즈 플랜츠 서비시즈, 인크. | Cas12a 뉴클레아제의 변이체 및 그의 제조 방법 및 용도 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015133554A1 (ja) * | 2014-03-05 | 2015-09-11 | 国立大学法人神戸大学 | 標的化したdna配列の核酸塩基を特異的に変換するゲノム配列の改変方法及びそれに用いる分子複合体 |
WO2016072399A1 (ja) * | 2014-11-04 | 2016-05-12 | 国立大学法人神戸大学 | 脱塩基反応により標的化したdna配列に特異的に変異を導入する、ゲノム配列の改変方法、並びにそれに用いる分子複合体 |
WO2017070632A2 (en) * | 2015-10-23 | 2017-04-27 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
WO2017090761A1 (ja) * | 2015-11-27 | 2017-06-01 | 国立大学法人神戸大学 | 標的化したdna配列の核酸塩基を特異的に変換する、単子葉植物のゲノム配列の変換方法、及びそれに用いる分子複合体 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE60310202T2 (de) | 2002-01-23 | 2007-11-22 | The University Of Utah Research Foundation, Salt Lake City | Zielgerichtete chromosomale mutagenese mit zinkfingernukleasen |
JP2013128413A (ja) | 2010-03-11 | 2013-07-04 | Kyushu Univ | Pprモチーフを利用したrna結合性蛋白質の改変方法 |
DK3494997T3 (da) * | 2012-07-25 | 2019-12-02 | Broad Inst Inc | Inducerbare dna-bindende proteiner og værktøjer til genomperturbation samt anvendelser deraf |
WO2017009073A1 (de) | 2015-07-13 | 2017-01-19 | Huf Hülsbeck & Fürst Gmbh & Co. Kg | Türaussengriff für ein fahrzeug |
DK3348638T3 (da) | 2015-09-09 | 2023-02-13 | Univ Kobe Nat Univ Corp | Fremgangsmåde til at konvertere genomsekvens fra gram-positiv bakterie ved specifikt at konvertere nukleinsyrebase i tilsigtet dna-sekvens, og molekylkompleks anvendt dertil |
ES2902338T3 (es) | 2015-09-09 | 2022-03-28 | Univ Kobe Nat Univ Corp | Método para modificar una secuencia genómica que convierte específicamente una nucleobase de una secuencia de ADN diana, y complejo molecular utilizado en dicho método |
-
2017
- 2017-04-21 JP JP2018513234A patent/JP7001272B2/ja active Active
- 2017-04-21 ES ES17786061T patent/ES2919961T3/es active Active
- 2017-04-21 CA CA3021281A patent/CA3021281C/en active Active
- 2017-04-21 US US16/094,587 patent/US20200377910A1/en active Pending
- 2017-04-21 EP EP17786061.6A patent/EP3447139B1/en active Active
- 2017-04-21 CN CN202311476665.5A patent/CN117925730A/zh active Pending
- 2017-04-21 CN CN201780038364.0A patent/CN109312329B/zh active Active
- 2017-04-21 DK DK17786061.6T patent/DK3447139T3/da active
- 2017-04-21 SG SG11201809242VA patent/SG11201809242VA/en unknown
- 2017-04-21 BR BR112018071376-7A patent/BR112018071376A2/pt active Search and Examination
- 2017-04-21 KR KR1020187032638A patent/KR102116200B1/ko active IP Right Grant
- 2017-04-21 WO PCT/JP2017/016105 patent/WO2017183724A1/ja active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015133554A1 (ja) * | 2014-03-05 | 2015-09-11 | 国立大学法人神戸大学 | 標的化したdna配列の核酸塩基を特異的に変換するゲノム配列の改変方法及びそれに用いる分子複合体 |
WO2016072399A1 (ja) * | 2014-11-04 | 2016-05-12 | 国立大学法人神戸大学 | 脱塩基反応により標的化したdna配列に特異的に変異を導入する、ゲノム配列の改変方法、並びにそれに用いる分子複合体 |
WO2017070632A2 (en) * | 2015-10-23 | 2017-04-27 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
WO2017090761A1 (ja) * | 2015-11-27 | 2017-06-01 | 国立大学法人神戸大学 | 標的化したdna配列の核酸塩基を特異的に変換する、単子葉植物のゲノム配列の変換方法、及びそれに用いる分子複合体 |
Non-Patent Citations (6)
Title |
---|
KITAMURA K ET AL.: "Uracil DNA glycosylase counteracts APOBEC3G-induced hypermutation of hepatitis B viral genomes: excision repair of covalently closed circular DNA", PLOS PATHOG., vol. 9, no. 5, 16 May 2013 (2013-05-16), pages el003361, XP055432854, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pubmed/23696735> [retrieved on 20160710] * |
KOMOR AC ET AL.: "Programmable editing of a target base in genomic DNA without double- stranded DNA cleavage", NATURE, vol. 533, no. 7603, 20 April 2016 (2016-04-20), pages 420 - 424, XP055343871, Retrieved from the Internet <URL:http://www.nature.com/nature/journal/v533/n7603/full/nature17946.html> [retrieved on 20170706] * |
NISHIDA K ET AL.: "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems", SCIENCE, vol. 353, 4 August 2016 (2016-08-04), pages aaf8729, XP055367833, Retrieved from the Internet <URL:http://science.sciencemag.org/content/353/6305/aaf8729.1ong> [retrieved on 20170706] * |
ONTICELLO SG ET AL.: "Evolution of the AID/ APOBEC family of polynucleotide (deoxy)cytidine deaminases", MOL. BIOL. EVOL., vol. 22, no. 2, 2005, pages 367 - 377, XP002667007 * |
ROGOZIN IB ET AL.: "Evolution and diversification of lamprey antigen receptors: evidence for involvement of an AID-APOBEC family cytosine deaminase", NAT. IMMUNOL., vol. 8, no. 6, 2007, pages 647 - 656, XP002503283 * |
See also references of EP3447139A4 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020241869A1 (ja) * | 2019-05-30 | 2020-12-03 | 国立大学法人東京大学 | 2種の核酸塩基変換酵素が融合されたCasタンパク質を利用したゲノム編集システム |
Also Published As
Publication number | Publication date |
---|---|
JP7001272B2 (ja) | 2022-01-19 |
US20200377910A1 (en) | 2020-12-03 |
CN109312329A (zh) | 2019-02-05 |
CA3021281C (en) | 2021-07-27 |
DK3447139T3 (da) | 2022-07-04 |
CN109312329B (zh) | 2023-11-21 |
ES2919961T3 (es) | 2022-07-29 |
EP3447139A4 (en) | 2019-10-02 |
SG11201809242VA (en) | 2018-11-29 |
CN117925730A (zh) | 2024-04-26 |
CA3021281A1 (en) | 2017-10-26 |
KR102116200B1 (ko) | 2020-05-27 |
KR20180132862A (ko) | 2018-12-12 |
JPWO2017183724A1 (ja) | 2019-02-28 |
BR112018071376A2 (pt) | 2019-04-24 |
EP3447139A1 (en) | 2019-02-27 |
EP3447139B1 (en) | 2022-06-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11718846B2 (en) | Genomic sequence modification method for specifically converting nucleic acid bases of targeted DNA sequence, and molecular complex for use in same | |
JP6153180B2 (ja) | 脱塩基反応により標的化したdna配列に特異的に変異を導入する、ゲノム配列の改変方法、並びにそれに用いる分子複合体 | |
JP6780860B2 (ja) | 標的化したdna配列の核酸塩基を特異的に変換するゲノム配列の改変方法及びそれに用いる分子複合体 | |
WO2017183724A1 (ja) | ゲノム配列改変技術における変異導入効率の向上方法、及びそれに用いる分子複合体 | |
US20240117384A1 (en) | Method for converting nucleic acid sequence of cell specifically converting nucleic acid base of targeted dna using cell endogenous dna modifying enzyme, and molecular complex used therein | |
JP2020191879A (ja) | 細胞の有する二本鎖dnaの標的部位を改変する方法 | |
WO2019194320A1 (ja) | エンジニアリングされたBlCas9ヌクレアーゼ |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
ENP | Entry into the national phase |
Ref document number: 2018513234 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 3021281 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112018071376 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 20187032638 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2017786061 Country of ref document: EP |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17786061 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2017786061 Country of ref document: EP Effective date: 20181121 |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01E Ref document number: 112018071376 Country of ref document: BR Free format text: REAPRESENTE A DECLARACAO REFERENTE AO DOCUMENTO DE PRIORIDADE DEVIDAMENTE ASSINADA, CONFORME ART. 408 C/C ART. 410, II, DO CODIGO DE PROCESSO CIVIL. |
|
ENP | Entry into the national phase |
Ref document number: 112018071376 Country of ref document: BR Kind code of ref document: A2 Effective date: 20181017 |