JP7292258B2 - 縦列反復配列を有するドナーｄｎａ修復鋳型を使用する部位特異的なｄｎａ改変 - Google Patents
縦列反復配列を有するドナーｄｎａ修復鋳型を使用する部位特異的なｄｎａ改変 Download PDFInfo
- Publication number
- JP7292258B2 JP7292258B2 JP2020507491A JP2020507491A JP7292258B2 JP 7292258 B2 JP7292258 B2 JP 7292258B2 JP 2020507491 A JP2020507491 A JP 2020507491A JP 2020507491 A JP2020507491 A JP 2020507491A JP 7292258 B2 JP7292258 B2 JP 7292258B2
- Authority
- JP
- Japan
- Prior art keywords
- dna
- donor
- repair template
- sequence
- rca
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000033616 DNA repair Effects 0.000 title claims description 124
- 230000008836 DNA modification Effects 0.000 title description 10
- 108020004414 DNA Proteins 0.000 claims description 350
- 230000004048 modification Effects 0.000 claims description 75
- 238000012986 modification Methods 0.000 claims description 75
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 67
- 238000010362 genome editing Methods 0.000 claims description 67
- 239000002773 nucleotide Substances 0.000 claims description 64
- 125000003729 nucleotide group Chemical group 0.000 claims description 62
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 61
- 230000005782 double-strand break Effects 0.000 claims description 50
- 238000000034 method Methods 0.000 claims description 44
- 108020005065 3' Flanking Region Proteins 0.000 claims description 41
- 108091033409 CRISPR Proteins 0.000 claims description 39
- 230000003321 amplification Effects 0.000 claims description 38
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 38
- 108091028113 Trans-activating crRNA Proteins 0.000 claims description 31
- 108020005029 5' Flanking Region Proteins 0.000 claims description 27
- 108020005004 Guide RNA Proteins 0.000 claims description 17
- 230000008439 repair process Effects 0.000 claims description 15
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 claims description 12
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 claims description 11
- 238000005096 rolling process Methods 0.000 claims description 10
- 108091081062 Repeated sequence (DNA) Proteins 0.000 claims description 5
- 238000000338 in vitro Methods 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 239000000047 product Substances 0.000 description 97
- 210000004027 cell Anatomy 0.000 description 70
- 239000013612 plasmid Substances 0.000 description 68
- 108091079001 CRISPR RNA Proteins 0.000 description 42
- 239000000203 mixture Substances 0.000 description 41
- 238000001890 transfection Methods 0.000 description 39
- 238000010348 incorporation Methods 0.000 description 32
- 238000003752 polymerase chain reaction Methods 0.000 description 29
- 108090000623 proteins and genes Proteins 0.000 description 28
- 230000010354 integration Effects 0.000 description 26
- 238000006243 chemical reaction Methods 0.000 description 24
- 239000002585 base Substances 0.000 description 22
- 239000000872 buffer Substances 0.000 description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 238000002474 experimental method Methods 0.000 description 14
- 239000011541 reaction mixture Substances 0.000 description 14
- 102000053602 DNA Human genes 0.000 description 13
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 13
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 13
- 230000029087 digestion Effects 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 239000007983 Tris buffer Substances 0.000 description 10
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 230000037361 pathway Effects 0.000 description 9
- 102100039087 Peptidyl-alpha-hydroxyglycine alpha-amidating lyase Human genes 0.000 description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 8
- 108091028732 Concatemer Proteins 0.000 description 7
- 108060002716 Exonuclease Proteins 0.000 description 7
- 101710163270 Nuclease Proteins 0.000 description 7
- 229920001213 Polysorbate 20 Polymers 0.000 description 7
- 230000000295 complement effect Effects 0.000 description 7
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 7
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 7
- 102000013165 exonuclease Human genes 0.000 description 7
- 230000037431 insertion Effects 0.000 description 7
- 238000003780 insertion Methods 0.000 description 7
- 239000008188 pellet Substances 0.000 description 7
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 7
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 7
- 108091008146 restriction endonucleases Proteins 0.000 description 7
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 239000007995 HEPES buffer Substances 0.000 description 6
- 101001093116 Homo sapiens Protein transport protein Sec61 subunit beta Proteins 0.000 description 6
- 102100023093 Kalirin Human genes 0.000 description 6
- 102100036308 Protein transport protein Sec61 subunit beta Human genes 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 108010052305 exodeoxyribonuclease III Proteins 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 230000006780 non-homologous end joining Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000008685 targeting Effects 0.000 description 6
- 238000010361 transduction Methods 0.000 description 6
- 230000026683 transduction Effects 0.000 description 6
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 5
- 108010042407 Endonucleases Proteins 0.000 description 5
- 102000004533 Endonucleases Human genes 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 230000005484 gravity Effects 0.000 description 5
- 230000006801 homologous recombination Effects 0.000 description 5
- 238000002744 homologous recombination Methods 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000012096 transfection reagent Substances 0.000 description 5
- 108020004638 Circular DNA Proteins 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 108010007577 Exodeoxyribonuclease I Proteins 0.000 description 4
- 102100029075 Exonuclease 1 Human genes 0.000 description 4
- 239000012981 Hank's balanced salt solution Substances 0.000 description 4
- 101001003584 Homo sapiens Prelamin-A/C Proteins 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 102100026531 Prelamin-A/C Human genes 0.000 description 4
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 239000001103 potassium chloride Substances 0.000 description 4
- 235000011164 potassium chloride Nutrition 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000010354 CRISPR gene editing Methods 0.000 description 3
- 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 3
- 101150077556 LMNA gene Proteins 0.000 description 3
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 3
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 238000012239 gene modification Methods 0.000 description 3
- 230000009368 gene silencing by RNA Effects 0.000 description 3
- 230000005017 genetic modification Effects 0.000 description 3
- 235000013617 genetically modified food Nutrition 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 125000002652 ribonucleotide group Chemical group 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 230000005778 DNA damage Effects 0.000 description 2
- 231100000277 DNA damage Toxicity 0.000 description 2
- 102100022286 DNA repair-scaffolding protein Human genes 0.000 description 2
- 230000004568 DNA-binding Effects 0.000 description 2
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 2
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 101000825159 Homo sapiens DNA repair-scaffolding protein Proteins 0.000 description 2
- 238000009004 PCR Kit Methods 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 101150109492 SEC61B gene Proteins 0.000 description 2
- 101710176276 SSB protein Proteins 0.000 description 2
- 101710126859 Single-stranded DNA-binding protein Proteins 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 241000193996 Streptococcus pyogenes Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000003491 array Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000003508 chemical denaturation Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000003505 heat denaturation Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 239000013615 primer Substances 0.000 description 2
- 239000002987 primer (paints) Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- OSBLTNPMIGYQGY-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;boric acid Chemical compound OB(O)O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O OSBLTNPMIGYQGY-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108091092236 Chimeric RNA Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 230000008265 DNA repair mechanism Effects 0.000 description 1
- 230000007023 DNA restriction-modification system Effects 0.000 description 1
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 1
- 101710116602 DNA-Binding protein G5P Proteins 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 101710162453 Replication factor A Proteins 0.000 description 1
- 101710176758 Replication protein A 70 kDa DNA-binding subunit Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 241000320123 Streptococcus pyogenes M1 GAS Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 239000008051 TBE buffer Substances 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- OSQPUMRCKZAIOZ-UHFFFAOYSA-N carbon dioxide;ethanol Chemical compound CCO.O=C=O OSQPUMRCKZAIOZ-UHFFFAOYSA-N 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 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
- 239000001963 growth medium Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001915 proofreading effect Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000005382 thermal cycling Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc 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
- 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
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- Cell Biology (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US15/491,125 | 2017-04-19 | ||
| US15/491,125 US11834670B2 (en) | 2017-04-19 | 2017-04-19 | Site-specific DNA modification using a donor DNA repair template having tandem repeat sequences |
| PCT/US2018/028366 WO2018195313A1 (en) | 2017-04-19 | 2018-04-19 | Site-specific dna modification using a donor dna repair template having tandem repeat sequences |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| JP2020517299A JP2020517299A (ja) | 2020-06-18 |
| JP2020517299A5 JP2020517299A5 (enExample) | 2021-05-27 |
| JP7292258B2 true JP7292258B2 (ja) | 2023-06-16 |
Family
ID=62196680
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2020507491A Active JP7292258B2 (ja) | 2017-04-19 | 2018-04-19 | 縦列反復配列を有するドナーｄｎａ修復鋳型を使用する部位特異的なｄｎａ改変 |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US11834670B2 (enExample) |
| EP (1) | EP3612630B1 (enExample) |
| JP (1) | JP7292258B2 (enExample) |
| CN (1) | CN110709514B (enExample) |
| CA (1) | CA3062154A1 (enExample) |
| WO (1) | WO2018195313A1 (enExample) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3204496A1 (en) | 2014-10-10 | 2017-08-16 | Editas Medicine, Inc. | Compositions and methods for promoting homology directed repair |
| AU2015342749B2 (en) | 2014-11-07 | 2022-01-27 | Editas Medicine, Inc. | Methods for improving CRISPR/Cas-mediated genome-editing |
| CA2999500A1 (en) | 2015-09-24 | 2017-03-30 | Editas Medicine, Inc. | Use of exonucleases to improve crispr/cas-mediated genome editing |
| EP3433363A1 (en) | 2016-03-25 | 2019-01-30 | Editas Medicine, Inc. | Genome editing systems comprising repair-modulating enzyme molecules and methods of their use |
| EP4047092B1 (en) | 2016-04-13 | 2025-07-30 | Editas Medicine, Inc. | Cas9 fusion molecules, gene editing systems, and methods of use thereof |
| EP3652312A1 (en) | 2017-07-14 | 2020-05-20 | Editas Medicine, Inc. | Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites |
| US10350307B2 (en) * | 2017-09-18 | 2019-07-16 | General Electric Company | In vivo RNA or protein expression using double-stranded concatemeric DNA including phosphorothioated nucleotides |
| US20200370070A1 (en) * | 2017-11-17 | 2020-11-26 | The Johns Hopkins University | Compositions and methods for efficient genome editing |
| KR20210045360A (ko) | 2018-05-16 | 2021-04-26 | 신테고 코포레이션 | 가이드 rna 설계 및 사용을 위한 방법 및 시스템 |
| BR112022001230A2 (pt) * | 2019-07-23 | 2022-03-15 | Pioneer Hi Bred Int | Estratégia de design de doador para edição de genoma crispr-cas9 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016500262A (ja) | 2012-12-12 | 2016-01-12 | ザ・ブロード・インスティテュート・インコーポレイテッ | 遺伝子産物の発現を変更するためのＣＲＩＳＰＲ−Ｃａｓ系および方法 |
| US20160046985A1 (en) | 2013-03-15 | 2016-02-18 | Complete Genomics, Inc. | Multiple tagging of long dna fragments |
Family Cites Families (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5648245A (en) * | 1995-05-09 | 1997-07-15 | Carnegie Institution Of Washington | Method for constructing an oligonucleotide concatamer library by rolling circle replication |
| WO2009022656A1 (ja) * | 2007-08-10 | 2009-02-19 | Toto Ltd. | 組換え哺乳動物細胞、組換え哺乳動物細胞の製造方法、および目的タンパク質の生産方法 |
| US8921072B2 (en) | 2008-09-02 | 2014-12-30 | General Electric Compnay | Methods to generate DNA mini-circles |
| JP2013505013A (ja) * | 2009-09-18 | 2013-02-14 | セレクシス エス．エー． | 強化導入遺伝子発現およびプロセッシングの産物および方法 |
| US9273324B2 (en) * | 2010-12-05 | 2016-03-01 | Andrew S. Belmont | Recombinant gene expression |
| EP2718446A2 (en) | 2011-06-07 | 2014-04-16 | Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) | Improved recombination efficiency by inhibition of nhej dna repair |
| FI3597749T3 (fi) * | 2012-05-25 | 2023-10-09 | Univ California | Menetelmiä ja koostumuksia rna-ohjattua kohde-dna-modifikaatiota varten ja rna-ohjattua transkription modulaatiota varten |
| PT3363902T (pt) | 2012-12-06 | 2019-12-19 | Sigma Aldrich Co Llc | Modificação e regulação de genoma baseadas em crispr |
| EP4234696A3 (en) | 2012-12-12 | 2023-09-06 | The Broad Institute Inc. | Crispr-cas component systems, methods and compositions for sequence manipulation |
| US20150067922A1 (en) | 2013-05-30 | 2015-03-05 | The Penn State Research Foundation | Gene targeting and genetic modification of plants via rna-guided genome editing |
| WO2016021973A1 (ko) | 2014-08-06 | 2016-02-11 | 주식회사 툴젠 | 캄필로박터 제주니 crispr/cas 시스템 유래 rgen을 이용한 유전체 교정 |
| EP3204496A1 (en) | 2014-10-10 | 2017-08-16 | Editas Medicine, Inc. | Compositions and methods for promoting homology directed repair |
| AU2015342749B2 (en) | 2014-11-07 | 2022-01-27 | Editas Medicine, Inc. | Methods for improving CRISPR/Cas-mediated genome-editing |
| EP4335918A3 (en) * | 2015-04-03 | 2024-04-17 | Dana-Farber Cancer Institute, Inc. | Composition and methods of genome editing of b-cells |
-
2017
- 2017-04-19 US US15/491,125 patent/US11834670B2/en active Active
-
2018
- 2018-04-19 CA CA3062154A patent/CA3062154A1/en active Pending
- 2018-04-19 CN CN201880025960.XA patent/CN110709514B/zh active Active
- 2018-04-19 EP EP18725682.1A patent/EP3612630B1/en active Active
- 2018-04-19 JP JP2020507491A patent/JP7292258B2/ja active Active
- 2018-04-19 WO PCT/US2018/028366 patent/WO2018195313A1/en not_active Ceased
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016500262A (ja) | 2012-12-12 | 2016-01-12 | ザ・ブロード・インスティテュート・インコーポレイテッ | 遺伝子産物の発現を変更するためのＣＲＩＳＰＲ−Ｃａｓ系および方法 |
| US20160046985A1 (en) | 2013-03-15 | 2016-02-18 | Complete Genomics, Inc. | Multiple tagging of long dna fragments |
Non-Patent Citations (2)
| Title |
|---|
| Cell Reports，2016年，Vol.14，p.2263-2272 |
| Nucleosides, Nucleotides, and Nucleic Acids，2008年，Vol.27，p.224-243 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110709514A (zh) | 2020-01-17 |
| CN110709514B (zh) | 2024-03-08 |
| EP3612630B1 (en) | 2023-08-30 |
| US20180305718A1 (en) | 2018-10-25 |
| CA3062154A1 (en) | 2018-10-25 |
| US11834670B2 (en) | 2023-12-05 |
| JP2020517299A (ja) | 2020-06-18 |
| WO2018195313A1 (en) | 2018-10-25 |
| EP3612630A1 (en) | 2020-02-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7292258B2 (ja) | 縦列反復配列を有するドナーｄｎａ修復鋳型を使用する部位特異的なｄｎａ改変 | |
| US20230091847A1 (en) | Compositions and methods for improving homogeneity of dna generated using a crispr/cas9 cleavage system | |
| JP7605852B2 (ja) | クラスｉｉのｖ型ｃｒｉｓｐｒ系 | |
| CN111373041A (zh) | 用于基因组编辑和调节转录的crispr/cas系统和方法 | |
| EA038500B1 (ru) | Термостабильные нуклеазы cas9 | |
| US20200232005A1 (en) | Methods of Producing and Using Single-Stranded Deoxyribonucleic Acids and Compositions for Use in Practicing the Same | |
| KR102664627B1 (ko) | 포스포로티오에이트화 뉴클레오티드를 포함하는 이중 가닥 콘카테머 dna를 사용하는 생체내 rna 또는 단백질 발현 | |
| AU2020356441A1 (en) | A nucleic acid delivery vector comprising a circular single stranded polynucleotide | |
| KR20240107373A (ko) | C2c9 뉴클레아제 기반의 신규 게놈 편집 시스템 및 이의 응용 | |
| JP2024501892A (ja) | 新規の核酸誘導型ヌクレアーゼ | |
| US20230122979A1 (en) | Methods of sample normalization | |
| US20250059568A1 (en) | Class ii, type v crispr systems | |
| US20250084484A1 (en) | Methods and compositions for transcriptome analysis | |
| WO2024138131A1 (en) | Expanding applications of zgtc alphabet in protein expression and gene editing |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200326 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20210416 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20210416 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20220606 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220905 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20221219 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230309 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20230508 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20230606 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7292258 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |