CN111979262A - Plant transformation - Google Patents
Plant transformation Download PDFInfo
- Publication number
- CN111979262A CN111979262A CN202010512913.7A CN202010512913A CN111979262A CN 111979262 A CN111979262 A CN 111979262A CN 202010512913 A CN202010512913 A CN 202010512913A CN 111979262 A CN111979262 A CN 111979262A
- Authority
- CN
- China
- Prior art keywords
- fomv
- plant
- expression system
- sequence
- promoter
- 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.)
- Pending
Links
- 230000009466 transformation Effects 0.000 title description 13
- 241000710017 Foxtail mosaic virus Species 0.000 claims abstract description 157
- 241000196324 Embryophyta Species 0.000 claims abstract description 154
- 230000014509 gene expression Effects 0.000 claims abstract description 78
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 71
- 108091033409 CRISPR Proteins 0.000 claims abstract description 58
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 58
- 238000010362 genome editing Methods 0.000 claims abstract description 56
- 238000000034 method Methods 0.000 claims abstract description 50
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 37
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 32
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 32
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 32
- 239000002299 complementary DNA Substances 0.000 claims abstract description 23
- 239000002773 nucleotide Substances 0.000 claims abstract description 22
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 22
- 239000013603 viral vector Substances 0.000 claims abstract description 14
- 239000013598 vector Substances 0.000 claims description 57
- 230000003612 virological effect Effects 0.000 claims description 21
- 108010077850 Nuclear Localization Signals Proteins 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 15
- 241000589158 Agrobacterium Species 0.000 claims description 12
- 108091030071 RNAI Proteins 0.000 claims description 12
- 230000009368 gene silencing by RNA Effects 0.000 claims description 12
- 238000000338 in vitro Methods 0.000 claims description 12
- 230000008685 targeting Effects 0.000 claims description 11
- 241000700605 Viruses Species 0.000 claims description 9
- 239000002245 particle Substances 0.000 claims description 9
- 238000010367 cloning Methods 0.000 claims description 8
- 230000001404 mediated effect Effects 0.000 claims description 8
- 108700007698 Genetic Terminator Regions Proteins 0.000 claims description 7
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 7
- 239000003112 inhibitor Substances 0.000 claims description 7
- 238000012546 transfer Methods 0.000 claims description 7
- 108020005004 Guide RNA Proteins 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 5
- 101000760903 Foxtail mosaic virus Coat protein Proteins 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 230000003252 repetitive effect Effects 0.000 claims description 4
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 claims description 2
- 235000006008 Brassica napus var napus Nutrition 0.000 claims description 2
- 240000000385 Brassica napus var. napus Species 0.000 claims description 2
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 claims description 2
- 235000004977 Brassica sinapistrum Nutrition 0.000 claims description 2
- 241000701489 Cauliflower mosaic virus Species 0.000 claims description 2
- 229920000742 Cotton Polymers 0.000 claims description 2
- 244000068988 Glycine max Species 0.000 claims description 2
- 235000010469 Glycine max Nutrition 0.000 claims description 2
- 241000219146 Gossypium Species 0.000 claims description 2
- 241000238631 Hexapoda Species 0.000 claims description 2
- 240000005979 Hordeum vulgare Species 0.000 claims description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 claims description 2
- 206010021929 Infertility male Diseases 0.000 claims description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 2
- 208000007466 Male Infertility Diseases 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims description 2
- 235000007164 Oryza sativa Nutrition 0.000 claims description 2
- 240000003768 Solanum lycopersicum Species 0.000 claims description 2
- 235000021307 Triticum Nutrition 0.000 claims description 2
- 244000098338 Triticum aestivum Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 230000036579 abiotic stress Effects 0.000 claims description 2
- 230000023852 carbohydrate metabolic process Effects 0.000 claims description 2
- 235000021256 carbohydrate metabolism Nutrition 0.000 claims description 2
- 235000005822 corn Nutrition 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 230000004129 fatty acid metabolism Effects 0.000 claims description 2
- 230000002538 fungal effect Effects 0.000 claims description 2
- 230000002363 herbicidal effect Effects 0.000 claims description 2
- 239000004009 herbicide Substances 0.000 claims description 2
- 235000009566 rice Nutrition 0.000 claims description 2
- 238000010354 CRISPR gene editing Methods 0.000 claims 1
- 244000025254 Cannabis sativa Species 0.000 claims 1
- 235000014647 Lens culinaris subsp culinaris Nutrition 0.000 claims 1
- 244000043158 Lens esculenta Species 0.000 claims 1
- 240000004658 Medicago sativa Species 0.000 claims 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 claims 1
- 244000061176 Nicotiana tabacum Species 0.000 claims 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims 1
- 102000014450 RNA Polymerase III Human genes 0.000 claims 1
- 108010078067 RNA Polymerase III Proteins 0.000 claims 1
- 239000013256 coordination polymer Substances 0.000 claims 1
- 230000011890 leaf development Effects 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 3
- 108020004414 DNA Proteins 0.000 description 34
- 210000004027 cell Anatomy 0.000 description 28
- 210000001519 tissue Anatomy 0.000 description 28
- 102100033254 Tumor suppressor ARF Human genes 0.000 description 24
- 108010001545 phytoene dehydrogenase Proteins 0.000 description 19
- 239000013612 plasmid Substances 0.000 description 14
- 101710132601 Capsid protein Proteins 0.000 description 12
- 101710094648 Coat protein Proteins 0.000 description 12
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 12
- 101710125418 Major capsid protein Proteins 0.000 description 12
- 101710163270 Nuclease Proteins 0.000 description 12
- 101710141454 Nucleoprotein Proteins 0.000 description 12
- 101710083689 Probable capsid protein Proteins 0.000 description 12
- 208000015181 infectious disease Diseases 0.000 description 12
- 230000009261 transgenic effect Effects 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 9
- 230000009885 systemic effect Effects 0.000 description 8
- 108091028113 Trans-activating crRNA Proteins 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 230000008595 infiltration Effects 0.000 description 7
- 238000001764 infiltration Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 6
- 238000010453 CRISPR/Cas method Methods 0.000 description 6
- 101150108119 PDS gene Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 230000000670 limiting effect Effects 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 208000024891 symptom Diseases 0.000 description 6
- 230000001052 transient effect Effects 0.000 description 6
- 108091079001 CRISPR RNA Proteins 0.000 description 5
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 5
- 108700026244 Open Reading Frames Proteins 0.000 description 5
- 241000379147 Strawberry green petal phytoplasma Species 0.000 description 5
- 108091023040 Transcription factor Proteins 0.000 description 5
- 102000040945 Transcription factor Human genes 0.000 description 5
- 108700019146 Transgenes Proteins 0.000 description 5
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 5
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000030279 gene silencing Effects 0.000 description 5
- 238000012226 gene silencing method Methods 0.000 description 5
- 235000009973 maize Nutrition 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 241000209510 Liliopsida Species 0.000 description 4
- 241000209504 Poaceae Species 0.000 description 4
- 102000038627 Zinc finger transcription factors Human genes 0.000 description 4
- 108091007916 Zinc finger transcription factors Proteins 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000005782 double-strand break Effects 0.000 description 4
- 239000012636 effector Substances 0.000 description 4
- 241001233957 eudicotyledons Species 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000007480 sanger sequencing Methods 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 108091006106 transcriptional activators Proteins 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 3
- 241000207746 Nicotiana benthamiana Species 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 102000039471 Small Nuclear RNA Human genes 0.000 description 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 101150038500 cas9 gene Proteins 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000004624 confocal microscopy Methods 0.000 description 3
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000010363 gene targeting Methods 0.000 description 3
- 208000006278 hypochromic anemia Diseases 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 230000033001 locomotion Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000006780 non-homologous end joining Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 108091029842 small nuclear ribonucleic acid Proteins 0.000 description 3
- 125000006850 spacer group Chemical group 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- 230000028604 virus induced gene silencing Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108700001094 Plant Genes Proteins 0.000 description 2
- 241000710007 Potexvirus Species 0.000 description 2
- 244000062793 Sorghum vulgare Species 0.000 description 2
- 101900024951 Tomato bushy stunt virus RNA silencing suppressor p19 Proteins 0.000 description 2
- 241000714211 Turnip crinkle virus Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 235000021186 dishes Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012250 transgenic expression Methods 0.000 description 2
- 230000010474 transient expression Effects 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- PYNVSZMFFVWFQA-NOMGDLSISA-N 2-amino-9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(1-hydroxyethyl)oxolan-2-yl]-3h-purin-6-one Chemical compound O[C@@H]1[C@H](O)[C@@H](C(O)C)O[C@H]1N1C(NC(N)=NC2=O)=C2N=C1 PYNVSZMFFVWFQA-NOMGDLSISA-N 0.000 description 1
- OGHAROSJZRTIOK-KQYNXXCUSA-O 7-methylguanosine Chemical group C1=2N=C(N)NC(=O)C=2[N+](C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OGHAROSJZRTIOK-KQYNXXCUSA-O 0.000 description 1
- 206010001557 Albinism Diseases 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 101710151325 B2 protein Proteins 0.000 description 1
- 241000724306 Barley stripe mosaic virus Species 0.000 description 1
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- 108010040467 CRISPR-Associated Proteins Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000723655 Cowpea mosaic virus Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 241000208720 Nepenthes Species 0.000 description 1
- 208000009869 Neu-Laxova syndrome Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108091093105 Nuclear DNA Proteins 0.000 description 1
- 241000709992 Potato virus X Species 0.000 description 1
- 241000723762 Potato virus Y Species 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 240000005498 Setaria italica Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 241000710145 Tomato bushy stunt virus Species 0.000 description 1
- 244000111306 Torreya nucifera Species 0.000 description 1
- 235000006732 Torreya nucifera Nutrition 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 241000209149 Zea Species 0.000 description 1
- FHHZHGZBHYYWTG-INFSMZHSSA-N [(2r,3s,4r,5r)-5-(2-amino-7-methyl-6-oxo-3h-purin-9-ium-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl [[[(2r,3s,4r,5r)-5-(2-amino-6-oxo-3h-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl] phosphate Chemical compound N1C(N)=NC(=O)C2=C1[N+]([C@H]1[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=C(C(N=C(N)N4)=O)N=C3)O)O1)O)=CN2C FHHZHGZBHYYWTG-INFSMZHSSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229930014669 anthocyanidin Natural products 0.000 description 1
- 235000008758 anthocyanidins Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000010165 autogamy Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 235000019519 canola oil Nutrition 0.000 description 1
- 239000000828 canola oil Substances 0.000 description 1
- 230000036978 cell physiology Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- NWKFECICNXDNOQ-UHFFFAOYSA-N flavylium Chemical compound C1=CC=CC=C1C1=CC=C(C=CC=C2)C2=[O+]1 NWKFECICNXDNOQ-UHFFFAOYSA-N 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000008076 immune mechanism Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000012212 insulator Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 235000019713 millet Nutrition 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 231100001160 nonlethal Toxicity 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 235000002252 panizo Nutrition 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- -1 sgRNApds Proteins 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/00023—Virus like particles [VLP]
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/38011—Tombusviridae
- C12N2770/38022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention generally relates to methods and materials for gene or genome editing of plants. The present invention provides an expression system for expressing a complete Genome Editing (GE) system, such as CRISPR Cas9, for editing a target sequence in a plant, the expression system comprising: at least one nucleotide sequence comprising: (i) a promoter, (ii) a FoMV cDNA, wherein the promoter is operably linked to a FoMV cDNA encoding one or more recombinant foxtail mosaic virus (FoMV) viral vectors comprising at least one subgenomic promoter (SGP) operably linked to a heterologous nucleic acid encoding the GE system. The invention also provides for a FoMV RNA transcript suitable for editing said target.
Description
Technical Field
The present invention generally relates to methods and materials for gene or genome editing of plants.
Background
Gene targeting and Genome Editing (GE) systems hold great promise for specific gene targeting or precise genome editing, both for functional characterization of plant genes and for genetic improvement of crops.
GE systems typically involve the use of engineered nuclease systems. Nucleases can induce double-strand breaks (DSBs) or nicks in a target DNA sequence, such that repair of the break by non-homologous end joining or homology-directed repair can result in gene knock-out and/or insertion of the target sequence (targeted integration). Modulation and/or cleavage of endogenous genes can be achieved by using proteins and systems such as zinc finger protein transcription factors (ZFP-TFs), Zinc Finger Nucleases (ZFNs), transcriptional activators such as effector transcription factors (TALE-TFs), CRISPR/Cas transcription factors (see, e.g., Perez-Pinera et al (2013) Nature Methods 10: 973-976), transcriptional activators such as effector nucleases (TALENs), Ttago nucleases or using CRISPR/Cas systems in conjunction with engineered crRNA/tracr RNA ("single guide RNA") to direct specific cleavage.
In plants, one of the key challenges in inducing GE is to deliver the entire system (nuclease and guide) into the cell for efficient expression. This is usually achieved by time-consuming and laborious plant transformation of the whole or part of the system, but not for species that are difficult or recalcitrant to transform.
Transient kits (e.g., virus-induced gene silencing (VIGS)5,6Gene Complementation (VIGC)7Hekaihua (VIF)8,9) Has been widely used in plants and several RNA/DNA viruses have been previously used to express the sgRNA component of only the GE in Cas 9-transgenic plants10-12。
WO2014194190 relates to plant gene targeting and genome editing methods in the fields of molecular biology and genetic engineering of DNA molecules introduced on specific types of vectors.
Nevertheless, it can be seen that a general non-transgenic strategy that could be used to exploit GE technology in plant functional genomics and crop improvement would contribute to the art.
Disclosure of Invention
The present inventors have provided a non-transgenic ViGE system using foxtail mosaic virus (FoMV), which is a positive single-stranded RNA mosaic virus capable of infecting monocotyledons and dicotyledons, to simultaneously transiently express the GE system represented by Cas9, sgRNA and RNAi repressor p19, in order to edit a target gene in plants.
Although, as described above, Cas9 transgenic plants have been used in the art (see Plant and Cell Physiology 58.4(2017):643-649 to Kaya et al), it is believed that the present invention expresses Cas9 from Plant viral vectors for the first time as part of a complete transient GE expression system.
Accordingly, in one aspect, there is provided a plant expression system for expressing a complete Genome Editing (GE) system for editing a target sequence in a plant, the expression system comprising at least one nucleotide sequence comprising:
(i) a plant-active promoter operably linked to a FoMV cDNA;
(ii) (ii) a FoMV cDNA encoding one or more recombinant foxtail mosaic virus (FoMV) viral vectors comprising at least one subgenomic promoter (SGP) operably linked to a heterologous nucleic acid encoding the GE system, and a terminator sequence;
(iii) a terminator sequence.
The invention also provides a process for producing said expression system, a method of editing a target sequence in a plant tissue, an RNA transcript from said expression system, a virus or viral particle encapsulating the RNA transcript, a kit comprising said expression system, and a plant host cell, tissue or whole plant comprising said expression system.
Preferably, the FoMV vector comprises all sequences required for replication and movement in the plant. The cDNA may be within an expression cassette, where the cDNA encodes the entire FoMV RNA genome (see, e.g., fig. 1A), but includes a subgenomic promoter operably linked to the heterologous nucleic acid.
Thus, the FoMV vector of the expression system is preferably fully functional. Once infiltrated into plant cells, they replicate and form viral particles that migrate between cells and spread systemically, forming systemic infections even in non-infiltrated leaf tissue.
However, the present invention is based on the use of transient system expressions and therefore does not employ the stable integration of one or more elements of the GE system as in the prior art. Thus, the system can be used to generate "transgene-free" plant tissue.
In another aspect, the system is directed to the use of RNA transcripts that have been expressed outside of the plant, e.g., extracted in vitro from suitable plasmids or other nucleic acids. Accordingly, the present invention additionally provides an in vitro expression system for expressing one or more RNA transcripts collectively encoding a complete Genome Editing (GE) system for editing a target sequence in a plant, wherein the expression system comprises at least one nucleotide sequence comprising:
(i) a promoter operably linked to a FoMV cDNA;
(ii) FoMV cDNA encoding one or more recombinant foxtail mosaic virus (FoMV) viral vectors comprising at least one subgenomic promoter (SGP) operably linked to a heterologous nucleic acid encoding the GE system.
In another aspect, the system can use RNA transcripts that have been expressed outside of the plant, e.g., isolated in vivo from a suitable plasmid or other encoding nucleic acid in a suitable microorganism or other isolated cell. Accordingly, the present invention additionally provides an in vivo expression system for expressing one or more RNA transcripts collectively encoding a complete Genome Editing (GE) system for editing a target sequence in a plant, wherein the expression system comprises at least one nucleotide sequence comprising:
(i) a promoter suitable for use in a microorganism or isolated cell, operably linked to a FoMV cDNA;
(ii) (ii) a FoMV cDNA encoding one or more recombinant foxtail mosaic virus (FoMV) viral vectors comprising at least one subgenomic promoter (SGP) operably linked to a heterologous nucleic acid encoding the GE system, and a terminator sequence;
(iii) a terminator sequence.
In another aspect, one or more isolated RNA transcripts (e.g., produced from an expression system described herein) that collectively encode a complete Genome Editing (GE) system for editing a target sequence in a plant are provided that encode one or more recombinant foxtail mosaic virus (FoMV) viral vectors containing at least one subgenomic promoter (SGP) operably linked to a heterologous nucleic acid encoding the GE system.
Preferably, the heterologous nucleic acid further comprises an RNAi inhibitor, e.g., p19 or a derivative thereof.
Preferably, the GE system consists of: (a) cas9, and (b) a small guide rna (sgrna) for targeting the GE system to a target sequence. The sgRNA takes a target sequence as a target point, and the Cas9 nuclease cuts a DNA molecule.
Thus, the expression system of the invention may itself comprise a vector or plasmid suitable for expression in plants or in vitro. They may consist of first and second expression vectors comprising or encoding first and second FoMV viral vectors each comprising heterologous nucleic acid encoding a GE system, e.g., the first vector expressing a nuclease such as Cas9, the second vector expressing the sgRNA and optionally the inhibitor.
FoMV is a species of Potexvirus and possesses a broad host range including 56 grasses and at least 35 dicots (Paulsen and Niblett, 1977; Short and Davies, 1987; Petty et al, 1989).
FoMV consists of a sense-antisense single-stranded (ss) RNA genome with a 5 '-methylguanosine cap, a 3' -poly A tail and five major Open Reading Frames (ORFs) and a unique 5A gene (Robertson et al, 2000). The five major ORFs all encode a functional protein (Robertson et al, 2000).
"plant-active promoter" refers to a nucleotide sequence for transcription initiation of a DNA operably linked downstream (i.e., in the 3' direction on the sense strand of a double-stranded DNA).
"operably linked" refers to being linked as part of the same nucleic acid molecule, in a position and orientation suitable for initiating transcription from a promoter (or subgenomic or other promoter). A nucleic acid operably linked to a promoter is "under transcriptional initiation control" of the promoter.
GE system
GE systems typically involve the use of engineered nuclease systems. In particular, nucleases can induce double-strand breaks (DSBs) or nicks in a target DNA sequence, such that repair of the break by non-homologous end joining (NHEJ) or homology-directed repair (HDR) can result in gene knock-out and/or insertion of the target sequence (targeted integration). Modulation and/or cleavage of endogenous genes can be achieved by using proteins and systems such as zinc finger protein transcription factors (ZFP-TFs), Zinc Finger Nucleases (ZFNs), transcriptional activators such as effector transcription factors (TALE-TFs), CRISPR/Cas transcription factors (see, e.g., Perez-Pinera et al (2013) Nature Methods 10: 973-976), transcriptional activators such as effector nucleases (TALENs), Ttago nucleases or using CRISPR/Cas systems in conjunction with engineered crRNA/tracr RNA ("single guide RNA") to direct specific cleavage.
Since the specificity of CRISPR/Cas systems is based on nucleotide pairing rather than protein-DNA interactions, this approach may be simpler, more specific, more efficient, and useful for plant genome editing than existing ZFNs and TALEN systems. WO2014194190 …
Suppressor
Gene silencing suppressors useful in the present invention are known in the art and described in WO/2007/135480. Preferably, the inhibitor is tomato bushy stunt virus P19 or a mutant thereof.
Nucleic acids and vectors
The nucleic acids of the invention may be isolated and/or purified, in essentially pure or homogeneous form, or free or substantially free of other nucleic acids. The term "isolated" encompasses all of these possibilities.
The vector used in the present invention is a "ViGE vector", which means a vector suitable for delivering a gene editing system to a plant by replicating a viral transcript in the plant. These can be delivered by introduction into the expression system of the plant or directly by RNA transcripts. In general, those skilled in the art will be able to construct vectors according to the invention, whether for plants or for microbial cells or other isolated cells or expression systems, given the disclosure of the invention. In addition to the promoter, terminator, the vector may also include other regulatory sequences, for example to define an expression cassette consisting of a modified recombinant FoMV cDNA and a heterologous nucleotide sequence. For details, for example, Molecular Cloning: a Laboratory Manual, second edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. Many known techniques and Protocols for manipulating nucleic acids, such as preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and gene expression, and protein analysis, are described in detail in Protocols in Molecular Biology, Second Edition, Ausubel et al eds., John Wiley & Sons, 1992. Specific procedures and vectors which have previously achieved widespread success in plants are described by Bevan, Nucl. acids Res. (1984) 12, 8711-. See: plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148.
In one aspect of the invention, a microbial expression vector is based on a plasmid, such as pUC or pBluescript, that can be used to transform e.coli expressing T7 RNA polymerase.
In one aspect of the invention, a plant expression vector is based on a plant binary transformation vector, such as pYL44, a pBIN 19-based T vector containing repeats of the CaMV 35S promoter and NOS terminator therein (Liu et al, 2002 b; also materials and methods below).
Transfer sequence
In one embodiment, the plant expression system further comprises a border sequence allowing for transfer of the nucleotide sequence into the plant genome at the entry site, e.g. a border sequence extending from agrobacterium tumefaciens. In these embodiments, the nucleotide sequence is located between the border sequences and can be inserted into the plant genome under appropriate conditions. Typically, this can be achieved by using the so-called "agroinfiltration", a method that uses agrobacterium-mediated transient transformation. Briefly, this technique is based on the property of Agrobacterium tumefaciens to transfer a portion of its DNA ("T-DNA") into a host cell where it may be integrated into nuclear DNA. T-DNA is defined by a border sequence of about 25 nucleotides in length. In the present invention, the border sequence is included around a "transfer nucleotide sequence" (T-DNA), wherein the entire vector is introduced into the plant by agricultural infiltration, or may be in the form of a binary transformation vector.
However, such border sequences are not essential for the use of the invention, and are dependent on the replication and movement of the FoMV viral nucleic acid in the plant. Thus, such a border sequence may not exist.
Plant promoters
Suitable plant-active promoters are well known to those skilled in the art and include the cauliflower mosaic virus 35S (CaMV 35S) gene promoter expressed at high levels in virtually all plant tissues. The promoter may in principle be an inducible promoter, for example the maize glutathione-S-transferase isoform II (GST-II-27) gene promoter, which is activated in response to the administration of an exogenous safener (WO93/01294, ICI Ltd). The GST-II-27 gene promoter has been shown to be induced by certain compounds that are useful in growing plants. Another suitable promoter may be the DEX promoter (Plant Journal (1997)11: 605-.
In vitro synthesis of RNA transcripts
As an alternative to introducing expression systems into plants, the present invention may be practiced using in vitro or in vivo synthesis of single stranded FoMV RNA molecules.
The in vitro synthesis of single-stranded RNA molecules is a routine laboratory procedure. Modern commercially available multipurpose cloning vectors typically contain a Multiple Cloning Site (MCS) flanked on each side by multiple promoters for different polymerases (e.g., SP6, T7, and T3 phage RNA polymerases, e.g., available from ThermoFisher Scientific).
Plasmid templates are typically linearized with restriction enzymes to allow synthesis of a missing RNA transcript with a defined end.
Most eukaryotic mRNA molecules have either 5' 7-methylguanosine residues or a cap structure, both of which play a role in the initiation of protein synthesis, protecting the mRNA from intracellular nuclease digestion. Capped in vitro transcripts can be synthesized by replacing a portion of GTP in the transcription reaction with a cap analogue (m7G (5') ppp (5') G).
Thus, both in vitro and in vivo FoMV GE expression systems as well as FoMV GE RNA transcripts constitute the invention.
Subgenomic promoters (SGPs)
According to the ViGE vector of the present invention, the heterologous GE system nucleic acid is operably linked to a subgenomic promoter that is recognized by an efficient replicase and results in transcription of subgenomic RNA corresponding to all or part of the GE system.
In one embodiment, the SGP is a FoMV SGP, such as a coat protein. However, SGPs of other potato viruses such as potato virus x (pvx) (5'-gaacggttaagtttccattgatactcgaaaga-3') can be used in place of FoMV SGP to control expression of heterologous nucleic acids encoding the GE system.
The SGP linked to the heterologous nucleic acid encoding the GE system is typically an additional SGP introduced into the FoMV sequence or a repetitive (second) SGP based on the native SGP of the FoMV. The first subgenomic promoter operably linked to a heterologous sequence is preferably the same sequence as the second subgenomic promoter. In this example, the first and second SGPs may both be from the same FoMV ORF, e.g., they may both be Coat Protein (CP) SGPs or both may be Triple Gene Block (TGB) SGPs.
Thus, some aspects and embodiments herein are exemplified by an incoming copy (duplicate) of the FoMV CP SGP, which is 5' from the local CP SGP and ORF. However, given the present disclosure, one skilled in the art will appreciate that other subgenomic promoters may be used, for example, the subgenomic promoter from the Triple Gene Block (TGB) of FoMV.
For example, the two subgenomic promoters include the native FoMV coat protein subgenomic promoter. This can be represented by the 170-bp sequence shown in 5202 to 5371 of SEQ ID NO. 1, but it will be appreciated that shorter or longer versions may be used as well. Generally, promoters are 100 to 500 nucleotides in length.
Some specific preferred embodiments will now be discussed:
CRISPR/CAS9
a preferred GE system in the context herein is the "clustered regularly interspaced short palindromic repeats" (CRISPR)/CRISPR-associated protein (Cas) system1. This system is an adaptive immune mechanism found in bacterial and archaeal species that allows the host to fight pathogens, such as bacteriophages (Barrangou et al, science315,1709-1712 (2007); Marraffini, L.A).&Sontheimer,E.J.Science 322,1843-1845(2008)；Bhaya,D.,Davison,M.&Barrangou, R.Annual review of genetics 45, 273-; garneau, j.e. et al Nature 468,67-71 (2010)). The phage-derived 30bp DNA fragment was inserted into the CRISPR locus of the host cell and transcribed to CRISPR RNA (crRNA). These form complexes with trans-encoding rna (tracrrna) and CRISPR-associated (Cas) proteins, and the complexes introduce site-specific cleavage at DNA sites that match the crRNA sequence. This mechanism has been applied to eukaryotes1-4Specific and multiple GE in (1).
CRISPR-Cas9 is a type II CRISPR-Cas system. The CRISPR-Cas9 system of streptococcus pyogenes is used in the art as a simple and versatile tool for RNA-guided genome editing (RGE) in different organisms. In Cas 9-mediated RGE, a single or dual short RNA molecule (short guide RNA or sgRNA) directs Cas9 to target a desired DNA site for genome modification or transcriptional control. The sgRNA-Cas9 recognizes the target DNA by sgRNA-DNA pairing between the 5' terminal leader sequence of the sgRNA (called the sgRNA spacer) and one DNA strand (the complementary strand of the prototype spacer). Cas9 also requires the Presence of A Motif (PAM) adjacent to the original spacer in the target site after the sgRNA-DNA pairing region.
In the expression systems described herein, the Cas9 sequence may include FLAG and/or nuclear localization signals. SEQ ID No: a non-limiting example of a Cas9 sequence is shown in fig. 3, although variants of this Cas9 sequence as well as other GE systems can also be used within the invention described herein.
Compositions and METHODS FOR making and using CRISPR-Cas systems are described in U.S. patent No.8,697,359, entitled "CRISPR-CAS SYSTEMS AND METHODS FOR isolating EXPRESSION OF GENE PRODUCTS," which is incorporated herein by reference in its entirety.
CRISPR-cas9 plasmids used in plants are commercially available, for example from addge, see: www.addgene.org/criprpr/plant/.
U6 promoter & sgRNA
Production of sgRNAs is typically driven by promoters from small nuclear RNAs (snRNAs), such as U6 and U3 snRNAs (see, e.g., Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. multiple gene engineering using CRISPR/Cas systems science 2013; 339(6121): 819-.
SEQ ID No.4 shows a non-limiting example of a U6 promoter scaffold suitable for use in the present invention. The scaffold also encodes tracrRNA (trans-activating crRNA) that plays a role in maturation of sgrnas.
sgrnas typically comprise a targeting sequence that corresponds to a target sequence in a target gene and comprises PAM.
Suppressor
Gene silencing suppressors useful in the present invention are known in the art and described in WO/2007/135480. They include HcPro from potato virus Y, He-Pro from TEV, P19 from TBSV, rgsCam, B2 protein from FHV, small coat protein of CPMV and coat protein from TCV. Preferably, the inhibitor is P19 or a mutant thereof.
Preferably, the mutant is a mutant P19 RNAi inhibitor, having potent RNAi inhibitory activity but lacking pathogenic function 15. One non-limiting example is shown in SEQ ID NO 2. Other modified P19 mutants are described for example by Shi et al (2009) "Suppression of local RNA sizing is not present to promoter cell-to-cell movement of Turnip crinkle virus in Nicotiana benthamiana" Plant signalling & Behavior 4:1,15-22 or by Scholthof US 20100269220.
Recombinant FoMV cDNA
Any suitable FoMV strain that produces a replicating, infectious FoMV viral transcript can be used in the present invention.
Within the FoMV sequence, all native FoMV ORFs, i.e. the FoMV RNA-dependent RNA polymerase, triple gene blocks and coat protein, are preferably retained.
However, one or more sequences may be deleted if the nucleic acid is still available for the production of a replicated infectious transcript.
Preferably, the elements in the nucleotide sequence are in the listed 5 'to 3' sequences.
Preferably, the vector system is based on the FoMV/P19: sgRNA (targeting the desired endogenous gene) and FoMV/Cas9, as described herein, e.g., with reference to fig. 1.
For example, the expression system may include FoMV/P19: sgRNApds, in which the PDS sequence is replaced with a different target gene sequence, including a suitable PAM. SEQ ID No:1 contains HpaI/AscI sites to facilitate cloning of different target gene sequences.
Substantially homologous variants of this sequence are also included within the scope of the invention. In particular, vectors derived from these FoMV vectors and having the characteristics of those vectors (described herein) are also included.
Targeted genes
For ViGE vectors, the GE system will include a "targeting gene", typically within a guide RNA or the like. This corresponds to the sequence in the plant to be edited.
Typically, the targeting sequence may be derived from an endogenous plant nuclear gene or transgene.
The methods of the invention may comprise identifying a PAM in the complementary strand of the target gene of interest. The methods of the invention can include engineering a sgRNA to be complementary to a selected target, wherein the 5' end of the engineered sgRNA is adjacent to the PAM.
A non-limiting example of the U6 promoter scaffold encoding the sgRNA (in this case, the plant PDS gene) is shown in SEQ ID No. 5.
ViGE is particularly useful for studying gene function because it can be used to make precise changes to a particular gene, thereby providing information about the function of that gene in vivo. In such cases, the targeting sequence may not be known, but the method of the invention can be used to identify its specific phenotype.
An "endogenous" gene is a gene that is present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid can comprise the genome of a chromosome, mitochondrion, chloroplast or other organelle, or an episomal nucleic acid that occurs in nature. Other endogenous molecules may include proteins, such as transcription factors and enzymes.
For purposes of the present invention, "gene" includes the DNA region encoding the gene product (see below), as well as all DNA regions that regulate the production of the gene product, whether or not such regulatory sequences are contiguous with coding and/or transcribed sequences. Thus, genes include, but are not limited to, promoter sequences, terminators, translation regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, border elements, origins of replication, matrix attachment sites, and locus control regions.
Targeted genes include those that confer plant traits and therefore may require editing using ViGE. For example, specific genes in mature tomato to improve the processing and handling characteristics of harvested fruit; a gene involved in pollen formation which enables a breeder to reproducibly produce a male sterile plant from the F1 hybrid; genes involved in lignin biosynthesis that improve the quality of pulp made from plant vegetative tissue; genes involved in the production of anthocyanidins, which genes give rise to new flowers and flowers; genes involved in regulatory pathways controlling development or environmental responses, which produce plants with new growth habits or, for example, disease resistance; eliminating toxic secondary metabolites through gene silencing of genes required for toxin production.
The plant of the present invention has utility
The invention is particularly applicable to plants that are native hosts (compatible with FoMV).
By "compatible" is meant capable of operating with other components of the system, in which case the FoMV must be capable of being replicated in the plant in question. These include not only the plants used in the examples below, but also other known hosts in the grass family, other monocotyledonous and dicotyledonous plants13。
The plant may be a dicot or a monocot.
Foxtail millet is of the family Gramineae and is used for understanding grain crops and C4Model systems for functional genomics and photosynthesis in plants. In a preferred embodiment, the plant may thus be a gramineae and/or C4 plant.
Non-limiting examples of preferred plants include wheat, millet, barley, corn, soybean, rice, cotton, canola oil seed (canola, OSR), sugar cane, sugar beet, potato, sorghum, and sunflower.
Other aspects of the invention
One aspect of the invention is a process for producing a vector as described above, substantially as described in the examples below, e.g. by introducing the GE system nucleic acid into cDNA encoding the genome of FoMV, optionally together with an additional CP subgenomic promoter (SGP).
One aspect of the invention includes a method of using ViGE to edit a target sequence or gene in a plant or plant tissue, the method comprising the steps of: introducing into the plant a vector as described above, wherein the vector comprises a GE sequence as a targeting sequence, such that the GE system is expressed in cells in the plant or tissue. The editing can alter the sequence or expression of a gene product of a target sequence, wherein the target sequence is a target gene.
A "plant tissue" is any tissue of a plant in vivo or in culture, including organs, cuttings, or any group of plant cells organized into structural and functional units of the whole plant.
The plant tissue may be a part of an early plant, such as two or three plant stages.
The method is preferably useful for causing non-lethal ViGE of target genes in plants, e.g. associated with mosaic symptoms of only mild FoMV infection.
As mentioned above, for introduction into the plant, the vector may be in the form of an Agrobacterium binary vector. The vector is introduced into the plant cell by Agrobacterium-mediated T-DNA transfer, and the transfer sequence can be transiently integrated into the plant (cell) genome and subsequently transcribed from the plant promoter into RNA.
Further provided is a process comprising introducing the ViGE vector into a plant.
In another aspect of the invention, there is provided a method comprising causing or allowing transcription from one or more of the ViGE vectors disclosed herein within the genome of a plant cell to produce a viral particle.
The invention also discloses a method for characterizing a target gene, which comprises the following steps:
(a) using the FoMV ViGE system as described above to edit the target gene in a part or a certain growth stage of the plant;
(b) observing the phenotype of the part of the plant in which the target gene is edited.
Typically, the observations will be compared to plants expressing the gene of interest to determine the characteristics of the gene (i.e., to establish one or more phenotypic characteristics).
In another aspect, a method of altering the phenotype of a plant is disclosed, the method comprising using the above editing method. Characteristics that may require alteration of their phenotype include: plant growth status, herbicide tolerance, drought resistance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, improved seed yield, improved oil percentage, improved protein percentage, and resistance to bacterial, fungal, or viral diseases.
In another aspect of the invention, a host cell comprising an expression system (vector or encoded virus or viral particle) is disclosed. These may be plant cells or may be microbial (in particular bacterial, especially agrobacterium) cells.
In another aspect, a plant or plant tissue is disclosed, including plants or plant tissues transiently transformed with one or more vectors of the present invention.
The invention also provides plants comprising such host cells or tissues.
The present disclosure also encompasses a plant seed that has been transiently infected, wherein in the seed a target gene has been edited as described above. These seeds may not include the expression system themselves. The invention also includes progeny, clones, cell lines or cells of the edited plants described above, wherein the progeny, clones, cell lines or cells have the edited gene, but optionally the expression system itself may not be included.
In another aspect of the invention, RNA transcripts from the above expression systems are provided.
In another aspect of the invention, there is provided a virus or viral particle encapsulating an RNA transcript from an expression system as described above.
In another aspect of the invention, there is provided a kit comprising a vector as described above.
It will be appreciated by those skilled in the art that the nucleotide sequences defined or listed herein, whether derived from or heterologous to FoMV, need not be "wild type", but may optionally be variants (e.g. mutants or other variants, or substantially homologous derivatives) so long as their function is not reversed. For example, the function of the FoMV coat protein is to encapsulate the FoMV genome and allow it to move. Various ORFs of FoMV and their functions will be discussed later. Likewise, the p19 derivatives maintain the function of inhibiting gene silencing.
"substantially homologous" means that the sequence in question is at least about 70% or 80% identical, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% identical to the reference sequence. Identity may be at the level of the nucleotide sequence and/or the encoded amino acid sequence. Preferably, FASTA and FASTP are used for sequence comparison (see Pearson & Lipman, 1988.Methods in Enzymology 183: 63-98). The default matrix is preferably used to set the parameters as follows: gapopen (penalty for the first residue in the gap): -12 for protein/16 for DNA; gapext (penalty for other residues in the gap): -2 for protein/4 for DNA; word length of KTUP: 2 for protein/6 for DNA
Any sub-titles contained herein are for convenience only and should not be construed as limiting the disclosure in any way.
The invention will be further described with reference to the following non-limiting figures and examples. In view of these, other embodiments of the invention will be apparent to those skilled in the art.
The disclosures of all references cited herein are specifically incorporated by cross-reference as they may be used to practice the present invention by those skilled in the art.
Drawings
Figure 1A shows virus-delivered Cas9 and sgRNApds-induced PDS gene editing.
(a) FoMV-based Cas9, sgRNApds, and p19 expression vectors. FoMV/Cas9, FoMV/sgRNApds and FoMV/sgRNAnon were used to express Cas9(FLAG: NLS: Cas9: NLS) labeled at the N-and C-termini with 3xFLAG and NLS labels, sgRNA targeting PDS genes and empty sgRNA scaffolds, respectively. FoMV/P19: sgRNApds are expected to co-express the p19 RNAi repressor and sgRNApds. Sgrna sequences, PDS target regions and Protospacer Adjacent Motif (PAM), and the FoMV genome and regulatory elements (left and right borders LB and RB, cloning site, double 35S promoter 2x35S, and NOS terminator) are shown. Arrows indicate the native and repetitive Coat Protein (CP) subgenomic RNA promoter.
(b) After infection with FoMV/Cas9+ FoMV/P19: cas9mRNA was detected in young leaf tissue of 3 Nb plants of sgRNApds, but not in 3 mock-inoculated control groups. The position and size of the DNA ladder (M) are shown, as well as the position of the Cas9mRNA detected.
(c, d) FoMV-mediated whole-body ViGE was detected in young leaf tissue. Comparing the Wild Type (WT) and 17 edited PDS sequences (P1 to P17), various point mutations and 2nt deletions were found in the sgRNApds target region (box, c). The representation of the chromatograms shows sgRNApds targeting sequences with ViGE mediated substitutions (asterisks) and deletions (2 triangles) (P1, P4, P13 and P16). (d) In that respect
(e) Various types and occurrences of ViGE events in sgRNApds-targeted PDS genes are summarized. PAM highlights (c, d), position number of PAM upstream nucleotide (c, e).
Fig. 1B shows an in vitro system for producing RNA transcripts encoding the FoMV vectors for delivering Cas9 and sgRNApds.
Figure 2 shows the effective ViGE by co-delivery of Cas9, sgRNApds and p19 from FoMV.
(a-d) ViGE Induction. The experimental strategy (a) is outlined. Under vacuum pressure (b), the cells were purified with a probe carrying either FoMV/Cas9 or FoMV/P19: mixed agrobacteria of sgRNApds infiltrated broken Seed Coat (SC) seeds. Systemic viral symptoms (c, d) appear in Nb plants grown from agroinfiltrated seeds. Plants were photographed on day 2 seeds (b) spread on water soaked filter paper and on days 28(c) and 60(d) after Agrobacterium seed drench.
(e) P19 mRNA was analyzed. Expression of p19 was detected in virus-infected Nbs (lanes 1 and 2) but not in mock plants (lane 3). The position and size of the DNA ladder (lane M) are shown.
(f) Delivery of p19 from FoMV enhanced viral co-expression of Cas9 protein in plants. In the case of a hybrid formed by FoMV/Cas9+ FoMV/P19: in sgRNApds infected Nb young leaf tissue (lane 5) and p19 transgenic young leaf tissue infected with FoMV/Cas9+ FoMV/sgRNApds (lane 6), Cas9 to be 160-KDa FLAG tagged (asterisk) was detected by Western blot, but not in Nb young leaf tissue infected with FoMC/Cas9+ FoMC/sgRNA pds (lanes 1 and 2) or mock controls (lanes 3 and 4). The position and size of the protein marker (lane M) are shown.
(g, h) FoMV mediated ViGE in young leaf tissue of the system. A comparison (g) of Wild Type (WT) and 10 edited PDS sequences (P1 to P10) is shown. Representations of the chromatograms (P1, P3, P9 and P10) show nucleotide substitutions (asterisks) in the sgRNApds target sequence (h). PAM highlights (g, h).
FIG. 3 shows transient expression of eGFP from FoMV/eGFP.
(a) FoMV-based eGFP expression vector. The organization of the FoMV genome is shown, and the arrows indicate the native and repetitive Coat Protein (CP) subgenomic RNA promoter.
(b-c) confocal microscopy analysis of eGFP expression in Agrobacterium-infiltrated Nb leaves by FoMV/eGFP. Brightfield image of Nb epidermal cells (b), eGFP expression in green channel (c) and merged image of panels b and c (d). (c) Test strips were 100 microns.
Fig. 4 shows ViGE in Nb.
(a-d) Nb infection with recombinant FoMV. There were no obvious viral symptoms in plants infiltrated with either FoMV/sgRNAnon (a), FoMV/sgRNAnon + FoMV/Cas9(b), FoMV/sgRNApds (c), or FoMV/sgRNApds + FoMV/Cas9 (d). Photographs were taken 21 days after agrobacterium infiltration of the leaves.
(e, f) genomic PCR/MlyI analysis of ViGE. Agarose gel analysis of 6 representative samples (Nb infected with FoMV/sgRNApds + FoMV/Cas 9) in two separate experiments Expt I (e) and Expt II (f) showed that the sgRNApds target PCR product was fully sensitive to MlyI cleavage, indicating that no ViGE occurred in the FoMV-infected plants. Similar results were obtained with repeated experiments.
(g, h) cloning/sequencing analysis of ViGE. MlyI digested genomic PCR products from one Randomly Selected (RS) sample of Expts I and II were isolated from gel sections (box, g) and cloned into T-vector. Sanger sequencing plasmid DNA extracted from randomly selected colonies further confirmed that no mutation (h) was introduced in the sgRNApds target PDS sequence. The position and size (e-g) of the DNA ladder is shown. The corresponding regions of the wild-type (WT) PDS gene and sgRNApds target PDS sequences in these expected ViGE samples (P1 to P9) are identical (h). The MlyI site is indicated and the sgRNApds target PDS sequence region is underlined. PAM (h) is highlighted.
FIG. 5 shows potential FoMV infection in the Sum nepenthes
(a) Seeded Nb was simulated. (b) FoMV infected Nb. (c) RT-PCR detection of FoMV. FoMV was detected in whole-body young (i.e., not agroinfiltrated) leaf tissue of three representative plants (P1, P2, and P3) infiltrated with FoMV, but not in mock-controls (P1 and P2). All plants remained healthy and showed no mosaic, chlorosis or leaf curl characteristic of viral infection. Plants were photographed 21 days after agrobacteria leaf infiltration (a, b). The position and size (c) of the DNA ladder is shown.
Fig. 6 shows whole body ViGE in p19 transgenic Nb.
(a) Binary vector pEAQ-HT15P 19-transgenic expression cassette in (1). Nb was transformed with Agrobacterium tumefaciens LBA4404 carrying the pEAQ-HT vector. Five single copy homozygous lines were obtained.
(b) Representative of the healthy p19 transgene. (c) And (4) healthy Nb.
(d, e) severe mosaic, chlorosis and curliness in plants occurred from two independent p19 transgenic lines infected with FoMV/Cas9+ FoMV/sgRNApds. Photographs were taken 21 days after agrobacterium infiltration of the leaves.
(f) Genomic PCR/MlyI analysis of whole body ViGE. The experimental outline of the genomic PCR/MlyI analysis of ViGE in p19 transgenic Nb is shown. Genomic PCR was performed using primers PDS _ MLY ID-F3 and PDS _ Mly _ ID-R (supplementary Table 1), and the resulting product (407bp) was treated with MlyI. Partial or complete digestion indicated that ViGE occurred in p19 transgenic Nb, but not in normal Nb infected with FoMV/Cas9+ FoMV/sgRNApds. The MlyI insensitive PCR product (e.g., triangle (f) here) was purified from agarose gel and cloned into a T-vector and sequenced. The position and size (f) of the DNA ladder is shown. It should be noted that in these ViGE plants there were no albinism or dwarfing phenotypes (d, e and FIGS. 2c, d), probably because most of the mutations were point mutations (FIGS. 2g, h), which did not result in changes in the amino acid sequence of the PDS protein.
Fig. 7 shows a systemic ViGE with co-delivery of Cas9, sgRNApds and p19 mediated by FoMV.
(a) Overview of the second method of ViGE detection. Genomic PCR was performed using the primers PDS _ MLY ID-F3 and PDS _ Mly _ ID-R (supplementary Table 1), and the resulting PCR product was directly cloned into a T-vector. After colony PCR screening and MlyI treatment of colony PCR products, Sanger sequencing was performed on miniprep plasmid DNA of MlyI insensitive samples.
(b, c) MlyI-treated agarose gel analysis of colony PCR products. In two separate ViGE experiments, pair 63
(b) And 26(c) colony PCR products were processed (indicated by diagonal lines) or not using MlyI. Those sequences with the expected size (407bp) and that were completely resistant to MlyI digestion (asterisk) were selected for sequencing (FIG. 2g, h).
Detailed Description
Example 1 ViGE Using FoMV vectors without Gene silencing
To test ViGE, we utilized originally VIGS13FoMV vectors designed but more recently directed to VIF14And transient gene expression was modified (FIG. 3). We cloned the coding sequence of 3xFLAG and Cas9 labeled with a Nuclear Localization Signal (NLS), Phytoene Desaturase (PDS) -targeted sgrna, or sgrna (sgrna) lacking any targeting sequence, into FoMV and the resulting FoMV/Cas9, FoMV/sgrna, and FoMV/sgrna, respectively.
Nicotiana benthamiana (Nb) plants infected with either FoMV/Cas9 in combination with either FoMV/sgRNAnon or FoMV/Cas9+ FoMV/sgRNApds did not show any symptoms by leaf coingregation, consistent with the detection of potential FoMV infection of viral RNA by RT-PCR (fig. 5 a-c). Then, we extracted genomic DNA from whole body leaf tissue and amplified sgRNApds target PDS gene. Complete MlyI digestion and subsequent sequencing analysis of the resulting PCR products showed that ViGE did not occur (FIGS. 4 e-h). We suspect that the initial failure of ViGE may be due to low efficacy of FoMV infection and insufficient viral expression of Cas9 and sgRNApds in plants.
Example 2 ViGE Using FoMV vectors with transiently expressed p19 repressor
To enhance the infectivity of FoMV to increase the levels of Cas9 and sgRNApds, we co-expressed tomato bushy stunt virus p19, a mutated RNAi repressor with strong RNAi inhibitory activity but no pathogenesis in plants 15. I generated a single copy homozygous Nb line transformed with the p19 expression cassette (FIG. 6 a). Transgenic plants infected with FoMV/sgRNApds and FoMV/Cas9 exhibited mosaic, chlorosis, and leaf curls (FIGS. 6b-e), and viral expression of Cas9mRNA was readily detected. Genomic PCR-MlyI screening of 24 infected p19 transgenes in 4 different experiments showed that sgRNApds target was only cleaved by MlyI, indicating that plant systemic ViGE occurred (fig. 6 f). Further cloning and sequencing of the PCR product against MlyI identified 104 mutations, including nucleotide deletions and substitutions in the target region. These findings are supported by the finding that GE content is increased in RNAi pathway-deficient Arabidopsis thaliana16。
Example 3 multifunctional transient whole body ViGE System for plants
Then, we generated a new vector, FoMV/P19: sgRNApds to express p19 and sgRNApds. With FoMV/Cas9 and FoMV/P19: after agroagulation of germinated Nb seeds by sgRNApds, plants grew and developed significant systemic viral symptoms (fig. 2 a-d). Viral delivery of p19 significantly enhanced the level of Cas9 protein in young leaf tissues (fig. 2e, f) and resulted in systemic ViGE effective against PDS targets (fig. 2g, h and fig. 7).
Example 4 use of the ViGE System in monocotyledons
Examples 1 to 3 illustrate how ViGE can be used effectively in dicotyledonous plants. The host range of the FoMV virus includes monocotyledonous and dicotyledonous plants, and therefore the invention is equally applicable to monocotyledonous plants.
The vector system described in example 2 (FoMV/Cas9 and FoMV/P19: sgRNAxxx) was introduced into tissues from maize (Zea maysL) using known techniques of transient expression, in which the sgRNA is targeted to the desired endogenous gene. Alternatively, RNA transcripts prepared by in vitro transcription were introduced into Maize tissue by bombardment (see, e.g., Yadava P, Abhishek A, Singh R et al Advances in Maize Transformation Technologies and Development of Transgenic Maize Sci.2017; published 7:1949.2017, 1/6.d., doi: 10.3389/fpls.2016.01949). Once the transcript penetrates into the plant cells, it replicates and forms viral particles that migrate between cells and spread systemically within the plant, establishing a systemic infection.
Conclusion of the examples
Combining our data indicates that simultaneous delivery of Cas9, sgRNA, and RNAi inhibitor p19 from FoMV results in the production of ViGE in plants. Expression of large proteins (e.g., Cas9) in excess of 160kD in size from FoMV for any plant virus-based gene expression system is also unprecedented17. This rapid and efficient method involves neither plant transformation nor transgenic expression of Cas9 or sgrnas. Considering the wide range of host species13,14FoMV-based ViGE is applicable to dicots and monocots, including important cereal crops.
Method of examples 1 to 3
Construction of a FoMV-based expression vector. Using high fidelity KOD-Plus-Neo DNA polymerase (Toyobo), as FLAG: NLS: Cas9: NLS4The coding sequence of 3xFLAG and Cas9 labeled with a Nuclear Localization Signal (NLS) (designated as FLAG: NLS: Cas9: NLS) was amplified using a set of primers Cas9-3X-NLS-Hpa-MLU-F and Cas9-3X-NLS-Xhol-ASC-R as template plasmids (supplementary Table 1). The resulting PCR product, approximately 4.2Kb in length, was then treated with HpaI and AscI and cloned into the binary vector pCambia2300-FoMV13To generate FoMV/Cas 9. For the production of FoMV/sgRNAnon and FoMV/sgRNApds, the high fidelity KOD-Plus-Neo DNA polymerase, pT-U6p-scaffold-U6t, was used11Or pCVA-gRNA as shown in the specification, NbPDS plasmid DNA11As a template and using primers AUT-Hpa-F3 and U6T-ASC-R2 (supplementary Table 1), HpaI was usedDigested with AscI and then cloned into the binary vector pCambia2300-FoMV13The corresponding DNA fragment was amplified from the HpaI/AscI site of (1). To generate FoMV/P19: sgRNApds, high fidelity KOD-Plus-Neo DNA polymerase was used, encoding the sequence P1915The P19 gene was amplified using the pEAQ-HT plasmid as a template and using a set of primers P19-ORF-F and P19-ORF-R (supplementary Table 1) and cloned into the Hpal site of FoMV/sgRNApds. Similarly, the eGFP gene was amplified using primers eGFP-ORF-F and eGFP-ORF-R (supplementary Table 1), plasmid pEGFP (Clontech) as template, and cloned into the Hpal site of pCambia2300-FoMV to produce FoMV/eGFP (FIG. 3 a). A pair of primers, Fomv Seq _6830_5K-F and Fomv Seq _7260_ SUBP-R (supplementary Table 1), was used to verify FLAG by PCR: NLS: cas9: NLS, sgRNAnon, sgRNApds, or P19: the insertion of sgRNApds in these FoMV vectors was further confirmed by Sanger sequencing. Prior to use in subsequent agroaortic infiltration experiments and plant transformation, by electroporation18All the FoMV constructs and the binary vector pEAQ-HT15Transformed into Agrobacterium tumefaciens LBA4404, respectively, and verified by sequencing of the plasmid prepared in minute quantities in Agrobacterium cultures.
Plant transformation such as19As described, a number of primary p 19-transgenic lines were generated by leaf disc transformation of Nicotiana benthamiana (Nb) with Agrobacterium tumefaciens LBA4404 containing pEAQ-HT. Specific primers P19-ORF-F and P19-ORF-R (supplementary Table 1) were used to verify transformation by PCR amplification of the integrated P19 transgene. After self-fertilization, the T1 and T2 progeny were tested for antibiotic sensitivity by germinating the seeds on 0.5mg/ml kanamycin. 5 independent single copy homozygous Nb lines transformed with the p19 transgene were obtained, with mendelian 3: 1 ratio of separation is demonstrated. Like WT Nb, all p19 transgenic plants grew properly and grew well.
Viral Infection (ViGE), plant growth, and maintenance. To prepare the FoMV and recombinant FoMV inocula, Agrobacterium tumefaciens LBA4404 carrying different FoMV constructs was cultured overnight in LB medium containing 0.5mg/ml streptomycin and 0.5mg/ml kanamycin (FIG. 3a) to reach 1.0OD at 28C600Is then made ofCollected by centrifugation at 3000rpm for 10 minutes and resuspended in sterile water to a final density of 0.5OD600. For agrobacterium-infiltrated leaves, agrobacterium is immersed in young leaves of wild-type or transgenic Nb plants at the six-leaf stage by 0.5 ml needle-free syringe. Alternatively, germinated seeds are subjected to agricultural infiltration to reduce viral infection time. Briefly, tens of Nb seeds were spread onto 3MM Whatman filter paper pre-soaked in sterile water and placed in petri dishes (10 cm diameter). The dishes were kept in the growth chamber under constant illumination at 25 ℃. After two days of standing under these conditions, the seeds began to dehull and germinate. At this stage, seeds were collected and stored in 50mL-FalconTMCentrifuge tube with 5ml 0.5OD600And (4) mixing the agrobacterium. Agroinfiltration of the seeds was achieved using a vacuum pump at a pressure of 0.085MPa for 10 minutes. The agroinfiltrated seeds were then transferred to compost and left in the dark for 24 hours. Subsequently, plants were grown in an insect-free growth chamber at 25 ℃ under 16 h light/8 h dark conditions, periodically checked for the presence of local and systemic infections, and recorded photographically using a D7000Sony NEX-5R camera.
Confocal microscopy. To examine eGFP expression from FoMV/eGFP, Nb leaves were harvested on day 4 after Agrobacterium infiltration and examined by Nikon A1 confocal microscopy at 488nm excitation to excite GFP and monitor emission of green fluorescence (510nm)20. Nb skins were also photographed in the bright field, as instructed by the manufacturer. Confocal images were processed using nikon a1 Nis-Elements software.
RNA extraction and RT-PCR. RNA was extracted from whole body Nb young leaf tissue using RNAprep pure plant kit (TIANGEN). First strand cDNA was synthesized from DNase I treated RNA (2. mu.g) by M-MLV reverse transcriptase using the FastQuant RT kit (TIANGEN) according to the manufacturer's instructions. PCR was performed to detect the FoMV genomic RNA using cDNA as a template along with either Cas9 or p19 mRNA expressed virally (supplementary table 1) with primers for each target and analyzed by 1.2% agarose gel electrophoresis.
Western blot analysis. Such as19Said young Nb leaves from the whole bodyTotal protein was extracted from the tissues. After electrophoresis at 100V for 2 hours, protein aliquots (20. mu.g) were separated on a 10% SDS-PAGE gel and transferred to nitrocellulose membrane (Bio-Rad). The following compositions were used: western blot analysis was performed with 2000 mouse anti-FLAG (Sigma-Aldrich) antibodies, run through a 1: 5000 goat anti-mouse IgG horseradish peroxidase conjugated secondary antibody (Abcam) and SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific). Chemiluminescence signals were detected using a ChemiDoc XRS + imaging system (Bio-Rad) as per the manufacturer's instructions.
Genomic DNA extraction and molecular characterization of ViGE. DNA was isolated from whole body Nb young leaf tissue using the DNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. Genomic PCR amplification of the sgRNA target PDS gene (407bp) was performed using high fidelity KOD-Plus-Neo DNA polymerase, 10-100ng of DNA as template and primers PDS _ MLY ID-F3 and PDS _ Mly _ ID-R (supplementary Table 1). Subsequently, two methods were used to characterize ViGE. In method I, genomic PCR products (about 400ng) were treated with MlyI (NEB) at 37 ℃ for 6 hours and analyzed by 1.5% agarose gel electrophoresis. Any undigested PCR fragment was purified from the gel and cloned into pEASY-Blunt3 cloning vector (TransGen Biotech) for Sanger sequencing. In method II, the genomic PCR product was cloned directly into the pEASY-Blunt3 cloning vector. After high fidelity colony PCR/MlyI digestion and screening, plasmid DNA is prepared in a trace manner for sequencing. It should be noted that the high fidelity KOD-Plus-Neo DNA polymerase and the detection of deletions and substitutions of various nucleotides including A → T and T → A ensure that these mutations we have identified are not the result of PCR errors, but are produced by the plant ViGE.
Supplementary Table 1
Reference to the literature
1.Jinek,M.et al.Science 337,816-821(2012).
2.Cong,L.et al.Science 339,819-823(2013)
3.Feng,Z.et al.Proc.Natl.Acad.Sci.USA 111,4632-4637(2014).
4.Yu,Z.et al.J.Integr.Plant Biol.60,376-381(2018).
5.Lin,Z.et al.Plant J.55,301-310(2008).
6.Chen,W.et al.Sci.Rep.5,9192(2015).
7.Zhou,T.et al.Sci.Rep.2,836(2012).
8.Li,C.et al.Sci.Rep.1,73(2011).
9.Qin,C.et al.Plant Physiol.174,875–885(2017).
10.Ali,Z.et al.Mol.Plant 8,1288-1291(2015).
11.Yin,K.et al.,Sci.Rep.5,14926(2015).
12.Cody,B.C.,Scholthof,H.B.&Mirkov,T.K.Plant Physiol.174,23–35(2017).
13.Liu,N.et al.Plant Physiol.171,1801-1807(2016)
14.Qin,C.et al.under review(2018).
15.Sainsbury,F.,Thuenemann,E.C.&Lomonossoff,G.P.Plant Biotechnol.J.7,682–693 (2009).
16.Mao,Y.et al.BMC Genome Biol.19,149(2018).
17.Kaya,H.,Kazuhiro Ishibashi,K.&Toki,S.Plant Cell Physiol.58,643–649(2017).
18.Chen et al.Plant Physiol.176,2700-2719(2018).
19.Hong,Y.,Saunders,K.,Hartley,M.R.&Stanley,J.Virology 220,119–127(1996).
20.Hong,Y.,Stanley,J.&van Wezel,R.J.Virol.77,13315–13322(2003).
Other references:
Paulsen A,Niblett C(1977)Purification and properties of foxtail mosaic virus.Phytopathology67:346-341
Petty I,Hunter B,Wei N,Jackson A(1989)Infectious Barley stripe mosaic virus RNA transcribed in Vitro from full-length genomic cDNA clones.Virology171:342-349
Short MN,Davies JW(1987)Host ranges,symptoms and amino acid compositions of eight potexviruses.Ann App Biol110:213-219
SEQ ID NO:1 FoMV vector sequences (6386nt, HpaI (5375) and AscI (5389) highlighted)
gaaaactcttccgaaaccgaaactgactgaaactacctcgaccgaccttagaacccaagaacccaacgggtgcggccacta tgtctatcgaggcagttttcgaccaggttacagacccatcgctccgcgctgtgattcaggaggaagcgcacaaacagatca aagatttgtttaaggaaacgacgcgctgcaatccctactccataccgcaagctgggcgcaaggttttggaaaagtacgcca tcccctacaacccgtactctctcaaactacaccctcacgcagcctcaaaagcgtttgaagtgtcgctctacgaggctgcgt ctaactacctcccctccacctcctcaactcctgtcacattcatgttcacaaaaccgggcaagctcagattctttaggcgcc gaggtcacgtggacaaattcgttaatgctgacatagttccaagagacttggctagatacccacgcgacacagtctacagtt atctgcccgagatcaccaccacacacgctttcattggcgacaccctacaccacttcggtgaggactttctcgtcgaggttt tctccaggtcaccgaaactagaagtgcttctagccaccatggtattaccacccgaggccttttacagaatggagtcccttc atccctcggtttacactctcctctacagggacgaccgattcctatacctgcctggtggcctgcctggcggtgagtacgaac atcgctataaggacctaaactggctaacatttggcacagttacgcacggcgggatcactatcacaggagaacgcattgaga ctaaggccgcgaatcatcttttcctcttcagacgagggcgactagagacaccaaaattccgctcattcgacatgcccgagc ctatggtcctgcttcccaaggttttccgccccgcaaagtacaatgtacagaagccaattccccgggagaaagcaaacaaat ggttgatgtacgttaaatccatcggcaatgccaccattcgtgacgtatgggctaagctgaggcaaaccatagccaatgcag acattggactcttctcgcccactgagctcgtgcatctcacgaattacttcctgctcctgggccggcttgactcacacaact ccttcgaccaagtactggccgacagtgtgctgaaagcatggttcagaccaatggtcgcaaagcttcaggagatcaagcaca aactcatggggcagacccaattcatgcaactctgccaagcgctagagatgacggaggtggacctcgtttttgaggtccggg actccaagactccccacaaacaagctgtgccgttggaccgtgaaattgaaaacgttttgttggaaggagtctcatcggagc caacttacacggaaaccgaaggcgttgctgatagtccacttccccccccaatgcaaactgcagccgagccgtccgcgacct cagacgagcccgagagctctagctcgcgtgaaattgagcaccaaccggcgcctgagatcacgcttgacgaggaagaacctc agcgagacgatctgccttgggacgcttggagaacacaattaagggcgcttggctttgaggcctctgaaaggcagtatgacc cggacggtgaactgatctctcctatcctgagcacccgaaggttacctaagactcccatagacacaacactctacgccacgc tagacaagattgcacgctgcccaactttctacaagcctgacacagatcgcgcgcagacttacgctcgcgatgtcatggcgg ggaaaaccggtgccattctcaagcaacaaccctttgagtggaaaaccacgctcaaacgcaagactaaagaggaaccgaagg aaattcaccttgcggtgttgcatggtgcgggcgggtcgggcaaatcctacgcactgcaggaatttatgcggaacaactctg acacaccgattacggtcatcctgccgactaacgagctcagggccgactggaagaaaaaattgcccgcccacgacaaagaca catttatgacatacgaaaacgcgctcttgtgccctcgtggagacatcttcattatggacgattacacaaaattgcccaggg gctacattgaggctttcgtgcagaatgcacctgccctctcacttctgatactcaccggtgaccccaaccaagccgaacact ttgagaccactgaggacaatgaaattaacagcctcgcccccgcctcagtggtcttcggcaagttctctaggtaccacataa atgccacacaccgcaaccccagaaacttggcaaacgccctcggtgtttactccgagacgcccggggaggttaaagtgcttt acacgaggaacatcaagaccggttatcacaatctcgtgccctcacaaatgaagatgagaaactacgcctcactcgggcagc gagcgtccacctatgcgggttgtcaggggatcactgcgccccgcgttcaaatcatcctagactccgacacaccccggtgca ccaggcaagtcatgtacactgcgctttcaagggccacgacggaagtggtgctctgcaacacgatgccggatgagaaaagct ttttccagaaggttgaagcaacaccgtacctcaaagccatcctcaacctcaacaaagagattaaagtcactgagggtgact tgacagaagaaccgccgagggagcccgctcctcccaccacacacctgcctgttgaaaacagaattattcttaatgaggccc tagtcgaaccgctgcccgacaaacatgaccgcgagatctactccaactccactggcttttcaaactgcatacagactcaag acccgtacatccaagccttccaacatcagcaagccaaggacgagacattgttctgggcaaccgtcgagaagaggctcgcag catccacgccgaaggacaactggacagaattcaagaccaagagacctctgggtgacgtgctttggctcgcgtacaagcggg cgatgatgctcccagatgagcccatcaaatttaacccagagctctggtgggcatgtgcagatgaggtgcaaaagacctacc tctccaagcccatacacgcgctcaagaacggaattcttcggcaatcacccgactttgactggaacaaactgcagattttcc tcaagtcacagtgggttaagaaaattgacaaaatcgggaaaattgacgtcaacgctggacagacgattgccgccttttacc aaccaaccgttatgctgtttggaaccatggcgagatacatgcgccgcatccgcgacacttatcaacccggcgaaatactca tcaattgcgagaagaaccagaagcacatttcgaagtgggtcgagagcaattggaaccaccgcctacccgcttacaccaatg acttcactgcttacgaccaaagccaggacggggctatgttacagtttgaggtactgaaagccctgcaccatgatatccctc atgaggttgtggaagcctacgtagcccttaaactcaactcaaaaatgtttctgggcacactggcgattatgagattaactg gtgagggacccaccttcgacgctaacacagagtgcaacattgcttacacacacgcccggttcgagatcccaaagaacgtgg cgcaaatgtacgcgggtgacgactgtgcgctcaactgcaggcccgttgaacggcagtccttcttgcctcttgtggagaaat tcaccctgaaatcaaaacccaaagtatttgagcaaaaagttgggtcatggcctgagttctgcggcaatctgatcaccccac ggggctacctcaaggatcccatgaagctacaacactgcctgcaactggcacagaggaagaaaccatccgaacctgggtcgc tcaaagacgttgctgagaactacgctatggacttgctacccacatacgagctaggtgatgcactctacgaaatcttcgacg agagacaaatgaacgcgcactatcagtcggtcaggacgcttatcacatgcgcccacaccaaagtcctccgagtggcacagg cacttcaggaagactgcaccttctttagctccatctaacaggttttgagttaggctaactccactgacgaattaaataaca atggatagtgaaatagttgaacgactaacaaagcttggtttcgtcaagacttcacacacgcacatcgctggcgagcccctc gtgattcacgccgttgctggggccggtaaaaccaccctccttcggtccttacttgaattaccgggcgtggaagtcttcaca ggcggggagcacgatcctccaaatttgtcagggaaatatatccgctgcgctgcaccccctgtggccggtgcatacaacatt ctcgacgagtaccccgcgtacccaaattggcgatcgcaaccctggaacgtcctaatcgccgacaacctacaatacaaagaa cccacagctcgcgcccactacacatgcaatcgcactcaccgcctggggcagctcactgtcgacgctttgcgtagggttggt ttcgacatcacctttgccggcacgcagactgaagactacggattccaggaaggccatctctacaccagtcaattttacgga caggtcatttcacttgacacgcaggcccataagatcgctgtgcgccacggacttgcacccctgtccgctttagaaacccgg gggctggaatttgatgagaccactgtgataacgactaaaacctcgctggaggaagtgaaggataggcacatggtctatgtc gctctcacacggcacaggcgcacctgccatctctacaccgctcactttgcgccctccgcctgacaacacgaaagccatact aactatagctataggaatagccgcctccctcgtctttttcatgctcacacgcaacaatctgccacacgtcggtgataacat ccactcactaccccacggaggaagttacattgacggtaccaagtccatcaactaccgcccacctgcgtcacgctacccctc atctaacttactcgctttcgctccaccaatactcgccgcagtgctctttttcctcacacagccatatctagctaccagacg atccaggtgcgttcggtgcttcgttgtccacggcgcatgcacgaatcacacctagttgttatattagcgctgttactttta gctctgtggtgtcttagcactcgacccgttcaaccatcgtgccatgtcgaaatcaacggccactccatcatcgtcaccgga aactgctggcactccactcaacgaccgcattgagggtgttagggtaaccaacatcagtgaagagaaacaacccacctcgag tgtgacctcatcatttcaggacacagttaacgcgtctcgaggcgcgccactcgacccgttcaaccatcgtgccatgtcgaa atcaacggccactccatcatcgtcaccggaaactgctggcactccactcaacgaccgcattgagggtgttagggtaaccaa catcagtgaagagaaacaacccacctcgagtgtgacctcatcatttcaggacacaatggcaccacaagatgccgacgtcac tgatgcgacggactacaagaaaccgcctgctgaaactgagcagaaggcactcaccattcaaccacggtcaaacaaggcgcc cagtgacgaggagttggtacgcatcatcaacgcggcgcagaagcgaggcctcacacccgcggcctttgttcaagcagctat agtcttcaccatggaatccatggacaagggcgccaccgactccacgattttcacgggaaaatacaacactttcccaatgaa aagtctggcgctagcttgcaaagatgctggcgtgcccgtgcacaaactttgctacttctataccaagccggcttacgcgaa ccgtagggtcgccaaccagccgcctgctcgctggaccaacgagaatgtgcccaaagctaacaagtgggcggctttcgacac cttcgacgcacttctcgacccatacgtagtcccatcctctgtaccgtacgatgagcccacgccagaggatcgccaagtcaa tgagattttcaagaaggacaatttgagtcaggcagcatccagaaaccaactccgcgccctaggaacgcaagcctccatcac gcgcgggagactcaacggcgcaccagcactaccaaacaacgggcagtacttcatcgaggcacctcagtgatcagtagtatg ataccaataaataaatcgggcgaatccgcgcctcctgactatgggcaggtttacggaccaagctgtatcgagatacgacct aacagtaacgcagctaaggggtgaatgcacacatcgcttataaaaaaaaaaaaaaaaaaaaaaaaaaa
2 mutant P19 sequence (the mutated nucleotides are underlined)
SEQ ID NO 3-3xFlag-Cas9-NLS (bold sequence: 3 xFlag; underlined sequence: NLS; oblique
Sequence in body notation: cas9)
SEQ ID NO 4-AtU6-Scaffold (AtU6 promoter: bold; tracrRNA, underlined; terminator, italic)
Body)
SEQ ID NO 5-AtU6-sgRNApds-Scaffold (AtU6 promoter: bold; sgRNApds: uppercase;
tracrRNA, underlined; terminator T, italic)
Sequence listing
<110> university of teachers in Hangzhou
<120> plant transformation
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6386
<212> DNA
<213> FoMV vector (FoMV)
<400> 1
gaaaactctt ccgaaaccga aactgactga aactacctcg accgacctta gaacccaaga 60
acccaacggg tgcggccact atgtctatcg aggcagtttt cgaccaggtt acagacccat 120
cgctccgcgc tgtgattcag gaggaagcgc acaaacagat caaagatttg tttaaggaaa 180
cgacgcgctg caatccctac tccataccgc aagctgggcg caaggttttg gaaaagtacg 240
ccatccccta caacccgtac tctctcaaac tacaccctca cgcagcctca aaagcgtttg 300
aagtgtcgct ctacgaggct gcgtctaact acctcccctc cacctcctca actcctgtca 360
cattcatgtt cacaaaaccg ggcaagctca gattctttag gcgccgaggt cacgtggaca 420
aattcgttaa tgctgacata gttccaagag acttggctag atacccacgc gacacagtct 480
acagttatct gcccgagatc accaccacac acgctttcat tggcgacacc ctacaccact 540
tcggtgagga ctttctcgtc gaggttttct ccaggtcacc gaaactagaa gtgcttctag 600
ccaccatggt attaccaccc gaggcctttt acagaatgga gtcccttcat ccctcggttt 660
acactctcct ctacagggac gaccgattcc tatacctgcc tggtggcctg cctggcggtg 720
agtacgaaca tcgctataag gacctaaact ggctaacatt tggcacagtt acgcacggcg 780
ggatcactat cacaggagaa cgcattgaga ctaaggccgc gaatcatctt ttcctcttca 840
gacgagggcg actagagaca ccaaaattcc gctcattcga catgcccgag cctatggtcc 900
tgcttcccaa ggttttccgc cccgcaaagt acaatgtaca gaagccaatt ccccgggaga 960
aagcaaacaa atggttgatg tacgttaaat ccatcggcaa tgccaccatt cgtgacgtat 1020
gggctaagct gaggcaaacc atagccaatg cagacattgg actcttctcg cccactgagc 1080
tcgtgcatct cacgaattac ttcctgctcc tgggccggct tgactcacac aactccttcg 1140
accaagtact ggccgacagt gtgctgaaag catggttcag accaatggtc gcaaagcttc 1200
aggagatcaa gcacaaactc atggggcaga cccaattcat gcaactctgc caagcgctag 1260
agatgacgga ggtggacctc gtttttgagg tccgggactc caagactccc cacaaacaag 1320
ctgtgccgtt ggaccgtgaa attgaaaacg ttttgttgga aggagtctca tcggagccaa 1380
cttacacgga aaccgaaggc gttgctgata gtccacttcc ccccccaatg caaactgcag 1440
ccgagccgtc cgcgacctca gacgagcccg agagctctag ctcgcgtgaa attgagcacc 1500
aaccggcgcc tgagatcacg cttgacgagg aagaacctca gcgagacgat ctgccttggg 1560
acgcttggag aacacaatta agggcgcttg gctttgaggc ctctgaaagg cagtatgacc 1620
cggacggtga actgatctct cctatcctga gcacccgaag gttacctaag actcccatag 1680
acacaacact ctacgccacg ctagacaaga ttgcacgctg cccaactttc tacaagcctg 1740
acacagatcg cgcgcagact tacgctcgcg atgtcatggc ggggaaaacc ggtgccattc 1800
tcaagcaaca accctttgag tggaaaacca cgctcaaacg caagactaaa gaggaaccga 1860
aggaaattca ccttgcggtg ttgcatggtg cgggcgggtc gggcaaatcc tacgcactgc 1920
aggaatttat gcggaacaac tctgacacac cgattacggt catcctgccg actaacgagc 1980
tcagggccga ctggaagaaa aaattgcccg cccacgacaa agacacattt atgacatacg 2040
aaaacgcgct cttgtgccct cgtggagaca tcttcattat ggacgattac acaaaattgc 2100
ccaggggcta cattgaggct ttcgtgcaga atgcacctgc cctctcactt ctgatactca 2160
ccggtgaccc caaccaagcc gaacactttg agaccactga ggacaatgaa attaacagcc 2220
tcgcccccgc ctcagtggtc ttcggcaagt tctctaggta ccacataaat gccacacacc 2280
gcaaccccag aaacttggca aacgccctcg gtgtttactc cgagacgccc ggggaggtta 2340
aagtgcttta cacgaggaac atcaagaccg gttatcacaa tctcgtgccc tcacaaatga 2400
agatgagaaa ctacgcctca ctcgggcagc gagcgtccac ctatgcgggt tgtcagggga 2460
tcactgcgcc ccgcgttcaa atcatcctag actccgacac accccggtgc accaggcaag 2520
tcatgtacac tgcgctttca agggccacga cggaagtggt gctctgcaac acgatgccgg 2580
atgagaaaag ctttttccag aaggttgaag caacaccgta cctcaaagcc atcctcaacc 2640
tcaacaaaga gattaaagtc actgagggtg acttgacaga agaaccgccg agggagcccg 2700
ctcctcccac cacacacctg cctgttgaaa acagaattat tcttaatgag gccctagtcg 2760
aaccgctgcc cgacaaacat gaccgcgaga tctactccaa ctccactggc ttttcaaact 2820
gcatacagac tcaagacccg tacatccaag ccttccaaca tcagcaagcc aaggacgaga 2880
cattgttctg ggcaaccgtc gagaagaggc tcgcagcatc cacgccgaag gacaactgga 2940
cagaattcaa gaccaagaga cctctgggtg acgtgctttg gctcgcgtac aagcgggcga 3000
tgatgctccc agatgagccc atcaaattta acccagagct ctggtgggca tgtgcagatg 3060
aggtgcaaaa gacctacctc tccaagccca tacacgcgct caagaacgga attcttcggc 3120
aatcacccga ctttgactgg aacaaactgc agattttcct caagtcacag tgggttaaga 3180
aaattgacaa aatcgggaaa attgacgtca acgctggaca gacgattgcc gccttttacc 3240
aaccaaccgt tatgctgttt ggaaccatgg cgagatacat gcgccgcatc cgcgacactt 3300
atcaacccgg cgaaatactc atcaattgcg agaagaacca gaagcacatt tcgaagtggg 3360
tcgagagcaa ttggaaccac cgcctacccg cttacaccaa tgacttcact gcttacgacc 3420
aaagccagga cggggctatg ttacagtttg aggtactgaa agccctgcac catgatatcc 3480
ctcatgaggt tgtggaagcc tacgtagccc ttaaactcaa ctcaaaaatg tttctgggca 3540
cactggcgat tatgagatta actggtgagg gacccacctt cgacgctaac acagagtgca 3600
acattgctta cacacacgcc cggttcgaga tcccaaagaa cgtggcgcaa atgtacgcgg 3660
gtgacgactg tgcgctcaac tgcaggcccg ttgaacggca gtccttcttg cctcttgtgg 3720
agaaattcac cctgaaatca aaacccaaag tatttgagca aaaagttggg tcatggcctg 3780
agttctgcgg caatctgatc accccacggg gctacctcaa ggatcccatg aagctacaac 3840
actgcctgca actggcacag aggaagaaac catccgaacc tgggtcgctc aaagacgttg 3900
ctgagaacta cgctatggac ttgctaccca catacgagct aggtgatgca ctctacgaaa 3960
tcttcgacga gagacaaatg aacgcgcact atcagtcggt caggacgctt atcacatgcg 4020
cccacaccaa agtcctccga gtggcacagg cacttcagga agactgcacc ttctttagct 4080
ccatctaaca ggttttgagt taggctaact ccactgacga attaaataac aatggatagt 4140
gaaatagttg aacgactaac aaagcttggt ttcgtcaaga cttcacacac gcacatcgct 4200
ggcgagcccc tcgtgattca cgccgttgct ggggccggta aaaccaccct ccttcggtcc 4260
ttacttgaat taccgggcgt ggaagtcttc acaggcgggg agcacgatcc tccaaatttg 4320
tcagggaaat atatccgctg cgctgcaccc cctgtggccg gtgcatacaa cattctcgac 4380
gagtaccccg cgtacccaaa ttggcgatcg caaccctgga acgtcctaat cgccgacaac 4440
ctacaataca aagaacccac agctcgcgcc cactacacat gcaatcgcac tcaccgcctg 4500
gggcagctca ctgtcgacgc tttgcgtagg gttggtttcg acatcacctt tgccggcacg 4560
cagactgaag actacggatt ccaggaaggc catctctaca ccagtcaatt ttacggacag 4620
gtcatttcac ttgacacgca ggcccataag atcgctgtgc gccacggact tgcacccctg 4680
tccgctttag aaacccgggg gctggaattt gatgagacca ctgtgataac gactaaaacc 4740
tcgctggagg aagtgaagga taggcacatg gtctatgtcg ctctcacacg gcacaggcgc 4800
acctgccatc tctacaccgc tcactttgcg ccctccgcct gacaacacga aagccatact 4860
aactatagct ataggaatag ccgcctccct cgtctttttc atgctcacac gcaacaatct 4920
gccacacgtc ggtgataaca tccactcact accccacgga ggaagttaca ttgacggtac 4980
caagtccatc aactaccgcc cacctgcgtc acgctacccc tcatctaact tactcgcttt 5040
cgctccacca atactcgccg cagtgctctt tttcctcaca cagccatatc tagctaccag 5100
acgatccagg tgcgttcggt gcttcgttgt ccacggcgca tgcacgaatc acacctagtt 5160
gttatattag cgctgttact tttagctctg tggtgtctta gcactcgacc cgttcaacca 5220
tcgtgccatg tcgaaatcaa cggccactcc atcatcgtca ccggaaactg ctggcactcc 5280
actcaacgac cgcattgagg gtgttagggt aaccaacatc agtgaagaga aacaacccac 5340
ctcgagtgtg acctcatcat ttcaggacac agttaacgcg tctcgaggcg cgccactcga 5400
cccgttcaac catcgtgcca tgtcgaaatc aacggccact ccatcatcgt caccggaaac 5460
tgctggcact ccactcaacg accgcattga gggtgttagg gtaaccaaca tcagtgaaga 5520
gaaacaaccc acctcgagtg tgacctcatc atttcaggac acaatggcac cacaagatgc 5580
cgacgtcact gatgcgacgg actacaagaa accgcctgct gaaactgagc agaaggcact 5640
caccattcaa ccacggtcaa acaaggcgcc cagtgacgag gagttggtac gcatcatcaa 5700
cgcggcgcag aagcgaggcc tcacacccgc ggcctttgtt caagcagcta tagtcttcac 5760
catggaatcc atggacaagg gcgccaccga ctccacgatt ttcacgggaa aatacaacac 5820
tttcccaatg aaaagtctgg cgctagcttg caaagatgct ggcgtgcccg tgcacaaact 5880
ttgctacttc tataccaagc cggcttacgc gaaccgtagg gtcgccaacc agccgcctgc 5940
tcgctggacc aacgagaatg tgcccaaagc taacaagtgg gcggctttcg acaccttcga 6000
cgcacttctc gacccatacg tagtcccatc ctctgtaccg tacgatgagc ccacgccaga 6060
ggatcgccaa gtcaatgaga ttttcaagaa ggacaatttg agtcaggcag catccagaaa 6120
ccaactccgc gccctaggaa cgcaagcctc catcacgcgc gggagactca acggcgcacc 6180
agcactacca aacaacgggc agtacttcat cgaggcacct cagtgatcag tagtatgata 6240
ccaataaata aatcgggcga atccgcgcct cctgactatg ggcaggttta cggaccaagc 6300
tgtatcgaga tacgacctaa cagtaacgca gctaaggggt gaatgcacac atcgcttata 6360
aaaaaaaaaa aaaaaaaaaa aaaaaa 6386
<210> 2
<211> 519
<212> DNA
<213> mutant P19(P19)
<400> 2
atggaacgag ctatacaagg aaacgacgct agggaacaag ctaacagtga acgttgggat 60
ggaggatcag gaggtaccac ttctcccttc aaacttcctg acgaaagtcc gagttggact 120
gagtggcggc tacataacga tgagacgaat tcgaatcaag ataatcccct tggtttcaag 180
gaaagctggg gtttcgggaa agttgtattt aagagatatc tcagatacga caggacggaa 240
gcttcactgc acagagtcct tggatcttgg acgggagatt cggttaacta tgcagcatct 300
cgatttttcg gtttcgacca gatcggatgt acctatagta ttcggtttcg aggagttagt 360
atcaccgttt ctggagggtc tcgaactctt cagcatctct gtgagatggc aattcggtct 420
aagcaagaac tgctacagct tgccccaatc gaagtggaaa gtaatgtatc aagaggatgc 480
cctgaaggta ctgagacctt cgaaaaagaa agcgagtaa 519
<210> 3
<211> 4269
<212> DNA
<213> Cas9 vector (Cas9)
<400> 3
atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat 60
gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc 120
gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 180
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 240
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 300
acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 360
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 420
gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 480
atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 540
ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 600
atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 660
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 720
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 780
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 840
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 900
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 960
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 1020
ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 1080
atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1140
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1200
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1260
aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1320
cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1380
attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1440
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1500
ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1560
gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1620
ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1680
aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1740
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1800
aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1860
ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1920
aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1980
accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 2040
ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 2100
ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2160
ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2220
ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2280
gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2340
aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2400
gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2460
aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2520
gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2580
atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2640
gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2700
aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2760
tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2820
aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2880
gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2940
aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 3000
ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 3060
caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3120
cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3180
atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3240
atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3300
ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3360
accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3420
acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3480
agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3540
tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3600
gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3660
ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3720
tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3780
aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3840
tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3900
cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3960
ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 4020
atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 4080
gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4140
gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4200
ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 4260
aagaaaaag 4269
<210> 4
<211> 527
<212> DNA
<213> U6 promoter (AtU6-Scaffold)
<400> 4
agcttcgttg aacaacggaa actcgacttg ccttccgcac aatacatcat ttcttcttag 60
ctttttttct tcttcttcgt tcatacagtt tttttttgtt tatcagctta cattttcttg 120
aaccgtagct ttcgttttct tctttttaac tttccattcg gagtttttgt atcttgtttc 180
atagtttgtc ccaggattag aatgattagg catcgaacct tcaagaattt gattgaataa 240
aacatcttca ttcttaagat atgaagataa tcttcaaaag gcccctggga atctgaaaga 300
agagaagcag gcccatttat atgggaaaga acaatagtat ttcttatata ggcccattta 360
agttgaaaac aatcttcaaa agtcccacat cgcttagata agaaaacgaa gctgagttta 420
tatacagcta gagtcgaagt agtggtttta gagctagaaa tagcaagtta aaataaggct 480
agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttt 527
<210> 5
<211> 546
<212> DNA
<213> U6 promoter (AtU6-sgRNApds-Scaffold)
<400> 5
agcttcgttg aacaacggaa actcgacttg ccttccgcac aatacatcat ttcttcttag 60
ctttttttct tcttcttcgt tcatacagtt tttttttgtt tatcagctta cattttcttg 120
aaccgtagct ttcgttttct tctttttaac tttccattcg gagtttttgt atcttgtttc 180
atagtttgtc ccaggattag aatgattagg catcgaacct tcaagaattt gattgaataa 240
aacatcttca ttcttaagat atgaagataa tcttcaaaag gcccctggga atctgaaaga 300
agagaagcag gcccatttat atgggaaaga acaatagtat ttcttatata ggcccattta 360
agttgaaaac aatcttcaaa agtcccacat cgcttagata agaaaacgaa gctgagttta 420
tatacagcta gagtcgaagt agtgccgtta atttgagagt ccagttttag agctagaaat 480
agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 540
tttttt 546
Claims (36)
1. A plant expression system for expressing a complete genome editing GE system for editing a target sequence in a plant, characterized in that the plant expression system comprises at least one nucleotide sequence comprising:
(i) a plant-active promoter operably linked to a FoMV cDNA;
(ii) (ii) a FoMV cDNA encoding one or more recombinant foxtail mosaic virus FoMV viral vectors comprising at least one subgenomic promoter SGP operably linked to a heterologous nucleic acid encoding the GE system, and a terminator sequence;
(iii) a terminator sequence.
2. The plant expression system of claim 1, wherein the plant promoter is a cauliflower mosaic virus 35S gene promoter or repetitive 35S promoter and/or the terminator sequence is a NOS terminator.
3. An in vitro expression system for expressing one or more RNA transcripts collectively encoding a complete genome editing GE system for editing a target sequence in a plant, characterized in that said expression system comprises at least one nucleotide sequence comprising:
(i) a promoter operably linked to a FoMV cDNA;
(ii) FoMV cDNA encoding one or more recombinant Fomv viral vectors of foxtail mosaic virus comprising at least one subgenomic promoter SGP operably linked to a heterologous nucleic acid encoding the GE system.
4. The expression system of any one of claims 1 to 3, characterized in that the heterologous nucleic acid further comprises an RNAi repressor.
5. The expression system of claim 4, wherein the RNAi inhibitor is p19 or a derivative thereof.
6. Expression system according to claim 5, characterized in that said p19 derivative is shown in SEQ ID No:2 in (c).
7. The expression system according to any one of claims 1 to 6, characterized in that the expression system comprises first and second expression vectors encoding first and second FoMV viral vectors, respectively, which together encode the GE system.
8. Expression system according to any one of claims 1 to 7, characterized in that the GE system comprises at least:
(a)Cas9；
(b) a small guide RNA, sgRNA, for targeting the GE system to the target sequence.
9. The expression system of claim 8, in which the Cas9 comprises FLAG and/or nuclear localization signal.
10. The expression system according to claim 8 or 9, characterized in that the sgRNA is operably linked to a DNA-dependent RNA polymerase III, Pol III, promoter sequence, which is an optional U6 promoter nucleotide sequence.
11. The expression system according to any one of claims 8 to 10, characterized in that the sgRNA includes a target sequence corresponding to a target sequence in a target gene and includes a protospacer adjacent motif PAM.
12. The expression system according to any one of claims 7 to 11, characterized in that the expression system comprises first and second FoMV viral vectors which together encode the GExit, and wherein the first vector expresses Cas9 and the second vector expresses the sgRNA and optionally the repressor.
13. The expression system according to any one of claims 1 to 12, characterized in that the FoMV cDNA comprises a first and a second FoMV SGP, wherein:
the first SGP is operably linked to the heterologous nucleic acid encoding the GE system, and
the second SGP subgenomic promoter is operably linked to its native FoMV ORF.
14. The expression system of claim 13, wherein the first and second CP subgenomic promoters comprise the same sequence, optionally the same sequence being the native FoMV coat protein subgenomic promoter.
15. The expression system of any one of claims 13 to 14, wherein the heterologous nucleic acid is located 5' to the second subgenomic promoter.
16. The expression system according to any one of claims 1 to 15, characterized in that the system comprises a FoMV vector as shown in SEQ ID No:1 into which a CRISPR sequence as shown in SEQ ID No:3 is inserted.
17. The expression system according to any one of claims 1 to 16, characterized in that the system comprises a FoMV vector as shown in SEQ ID No:1 into which an At U6 scaffold as shown in SEQ ID No:4 and a further sgRNA sequence have been inserted.
18. A process for producing the expression system according to any one of claims 1 to 17, characterized in that the process comprises:
(1) providing:
(i) a promoter operably linked to a FoMV cDNA;
(ii) a FoMV cDNA comprising at least one FoMV subgenomic promoter SGP operably linked to a multiple cloning site MCS;
(2) cloning a heterologous nucleic acid encoding the GE system into the MCS.
19. One or more isolated RNA transcripts, characterized in that said one or more isolated RNA transcripts, together encoding a complete genome editing GE system for editing target sequences in plants, optionally encoded by an expression system according to any one of claims 1 to 17, encode one or more recombinant foxtail mosaic virus FoMV viral vectors comprising at least one subgenomic promoter SGP operably linked to a heterologous nucleic acid encoding said GE system.
20. A method comprising introducing the plant expression system of any one of claims 1 to 17 or the isolated RNA transcript of claim 19 into plant tissue.
21. A method of editing a target sequence in a plant tissue, comprising the steps of: introducing the expression system according to any one of claims 1 to 17 or the isolated RNA transcript according to claim 19 into said plant tissue or an elite thereof.
22. The method of claim 21, wherein said editing alters the sequence or expression of a gene product of said target sequence, wherein said target sequence is a target gene.
23. The method of claim 22, wherein the gene product is characterized by one or more of the following: herbicide tolerance, drought resistance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, improved seed yield, improved oil percentage, improved protein percentage, and resistance to bacterial, fungal, or viral diseases.
24. Method according to any one of claims 20 to 23, characterized in that the system comprises a first and a second FoMV viral vector which together encode the GE system and optionally an RNAi repressor, and the two viral vectors are applied simultaneously.
25. The method according to any one of claims 20 to 24, wherein the plant tissue is a leaf or a seed, wherein the seed coat has been disrupted.
26. Method according to any one of claims 20 to 24, characterized in that said plant tissue is part of a plant at the 2 or 3 leaf development stage.
27. The method according to any one of claims 20 to 26, characterized in that the expression system is introduced by agrobacterium-mediated T-DNA transfer.
28. Method according to any one of claims 20 to 27, characterized in that said plant is a monocotyledonous plant.
29. Method according to claim 28, characterized in that said plant is selected from the list consisting of: tobacco, lentil, alfalfa, barley, wheat, grass, corn, rice, soybean, cotton, rape, canola, tomato, potato.
30. A method comprising providing progeny or seed of a elite plant, characterized in that a target sequence has been edited in a tissue of the elite plant according to the method according to any one of claims 20 to 29, wherein the progeny or seed comprise the edited target sequence.
31. A kit comprising the expression system according to any one of claims 1 to 17.
32. An isolated RNA transcript from the expression system of any one of claims 1 to 17.
33. A virus or viral particle encapsulating the RNA transcript according to claim 19 or 32.
34. A plant host cell comprising the expression system of any one of claims 1 to 17 or the transcript or virus or viral particle of claim 19, 32 or 33.
35. Plant tissue comprising or transiently transformed with the expression system of any one of claims 1 to 17 or the transcript or virus or viral particle of claim 19, 32 or 33.
36. A plant comprising the plant cell of claim 34 or the plant tissue of claim 35.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010512913.7A CN111979262A (en) | 2020-06-08 | 2020-06-08 | Plant transformation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010512913.7A CN111979262A (en) | 2020-06-08 | 2020-06-08 | Plant transformation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111979262A true CN111979262A (en) | 2020-11-24 |
Family
ID=73441997
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010512913.7A Pending CN111979262A (en) | 2020-06-08 | 2020-06-08 | Plant transformation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111979262A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024060538A1 (en) * | 2022-09-19 | 2024-03-28 | 中国科学院动物研究所 | Rice disease-resistant gene and use thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2651907A1 (en) * | 2006-05-22 | 2007-11-29 | Plant Bioscience Limited | Bipartite system, method and composition for the constitutive and inducible expression of high levels of foreign proteins in plants |
| CN103074310A (en) * | 2004-09-17 | 2013-05-01 | 先锋高级育种国际公司 | Isopentenyl transferase sequences and methods of use |
| CN107338264A (en) * | 2016-05-03 | 2017-11-10 | 清华大学 | The VIGS carriers of virus-mediated gene silencing |
-
2020
- 2020-06-08 CN CN202010512913.7A patent/CN111979262A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074310A (en) * | 2004-09-17 | 2013-05-01 | 先锋高级育种国际公司 | Isopentenyl transferase sequences and methods of use |
| CA2651907A1 (en) * | 2006-05-22 | 2007-11-29 | Plant Bioscience Limited | Bipartite system, method and composition for the constitutive and inducible expression of high levels of foreign proteins in plants |
| CN107338264A (en) * | 2016-05-03 | 2017-11-10 | 清华大学 | The VIGS carriers of virus-mediated gene silencing |
Non-Patent Citations (1)
| Title |
|---|
| 李田等: "植物启动子研究进展", 《生物技术通报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024060538A1 (en) * | 2022-09-19 | 2024-03-28 | 中国科学院动物研究所 | Rice disease-resistant gene and use thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gelvin | Integration of Agrobacterium T-DNA into the plant genome | |
| Kui et al. | Building a genetic manipulation tool box for orchid biology: identification of constitutive promoters and application of CRISPR/Cas9 in the orchid, Dendrobium officinale | |
| Iaffaldano et al. | CRISPR/Cas9 genome editing of rubber producing dandelion Taraxacum kok-saghyz using Agrobacterium rhizogenes without selection | |
| CN111373044A (en) | Methods of modifying the specificity of plant non-coding RNA molecules for silencing gene expression | |
| CN109196105B (en) | Methods of transfecting plants and reducing random integration events | |
| Zhang et al. | An RNAi suppressor activates in planta virus–mediated gene editing | |
| He et al. | Advances in gene editing without residual transgenes in plants | |
| US20190352653A1 (en) | Conferring resistance to geminiviruses in plants in alternative manner to gene drive, using crispr/cas systems | |
| US20230257762A1 (en) | Methods and compositions for plant pathogen resistance in plants | |
| Schmitz et al. | CRISPR/Cas9 mutagenesis by translocation of Cas9 protein into plant cells via the Agrobacterium type IV secretion system | |
| CN113811612A (en) | Production of dsRNA in several plant cells for pest control by gene silencing | |
| WO2018047183A1 (en) | Compositions and methods for regulating gene expression for targeted mutagenesis | |
| Golenberg et al. | Development of a gene silencing DNA vector derived from a broad host range geminivirus | |
| US7476780B2 (en) | Root agroinoculation method for virus induced gene silencing | |
| WO2019238772A1 (en) | Polynucleotide constructs and methods of gene editing using cpf1 | |
| Thomson et al. | Genomic consequences associated with Agrobacterium‐mediated transformation of plants | |
| CN111979262A (en) | Plant transformation | |
| AU2002256240B2 (en) | Ires enabled gene trapping in plants | |
| CN111630171A (en) | Lodging resistance of plants | |
| JP2024513588A (en) | Mobile endonucleases for inherited mutations | |
| AU2020274996B2 (en) | Modified plants comprising a polynucleotide comprising a non-cognate promoter operably linked to a coding sequence that encodes a transcription factor | |
| AU2002256240A1 (en) | Ires enabled gene trapping in plants | |
| Xu et al. | Transcriptome profiling reveals a petunia transcription factor, PhCOL4, contributing to antiviral RNA silencing | |
| Busov et al. | Transformation as a tool for genetic analysis in Populus | |
| WO2013154233A1 (en) | Sycmv-derived recombinant virus vector, and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201124 |