CN115851666B - Novel Cas enzymes and systems and uses - Google Patents
Novel Cas enzymes and systems and uses Download PDFInfo
- Publication number
- CN115851666B CN115851666B CN202211334825.8A CN202211334825A CN115851666B CN 115851666 B CN115851666 B CN 115851666B CN 202211334825 A CN202211334825 A CN 202211334825A CN 115851666 B CN115851666 B CN 115851666B
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- cas
- sequence
- protein
- target
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102000004190 Enzymes Human genes 0.000 title abstract description 20
- 108090000790 Enzymes Proteins 0.000 title abstract description 20
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 330
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 305
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 305
- 108090000623 proteins and genes Proteins 0.000 claims description 259
- 102000004169 proteins and genes Human genes 0.000 claims description 219
- 235000018102 proteins Nutrition 0.000 claims description 213
- 108020005004 Guide RNA Proteins 0.000 claims description 115
- 239000013598 vector Substances 0.000 claims description 76
- 238000003776 cleavage reaction Methods 0.000 claims description 68
- 230000007017 scission Effects 0.000 claims description 63
- 238000000034 method Methods 0.000 claims description 61
- 230000008685 targeting Effects 0.000 claims description 52
- 102000040430 polynucleotide Human genes 0.000 claims description 46
- 108091033319 polynucleotide Proteins 0.000 claims description 46
- 239000002157 polynucleotide Substances 0.000 claims description 46
- 239000000203 mixture Substances 0.000 claims description 41
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 33
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 30
- 230000027455 binding Effects 0.000 claims description 28
- 108020001507 fusion proteins Proteins 0.000 claims description 24
- 102000037865 fusion proteins Human genes 0.000 claims description 24
- 238000010362 genome editing Methods 0.000 claims description 20
- 230000001105 regulatory effect Effects 0.000 claims description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 15
- 241000282414 Homo sapiens Species 0.000 claims description 14
- 201000010099 disease Diseases 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 238000003745 diagnosis Methods 0.000 claims description 7
- 238000010363 gene targeting Methods 0.000 claims description 7
- 239000002243 precursor Substances 0.000 claims description 7
- 235000004252 protein component Nutrition 0.000 claims description 5
- 239000000969 carrier Substances 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 2
- 241000282412 Homo Species 0.000 claims 2
- 230000001225 therapeutic effect Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 41
- 101710163270 Nuclease Proteins 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 description 82
- 108020004414 DNA Proteins 0.000 description 54
- 230000000295 complement effect Effects 0.000 description 36
- 108091028043 Nucleic acid sequence Proteins 0.000 description 33
- 238000001514 detection method Methods 0.000 description 33
- 102000053602 DNA Human genes 0.000 description 31
- 238000006467 substitution reaction Methods 0.000 description 30
- 239000002773 nucleotide Substances 0.000 description 29
- 125000003729 nucleotide group Chemical group 0.000 description 29
- 239000013612 plasmid Substances 0.000 description 27
- 241000196324 Embryophyta Species 0.000 description 22
- 230000014509 gene expression Effects 0.000 description 22
- 239000000047 product Substances 0.000 description 22
- 150000001413 amino acids Chemical class 0.000 description 21
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 19
- 125000000539 amino acid group Chemical group 0.000 description 18
- 108090000765 processed proteins & peptides Proteins 0.000 description 17
- 238000007792 addition Methods 0.000 description 16
- 238000012217 deletion Methods 0.000 description 16
- 230000037430 deletion Effects 0.000 description 16
- 239000012634 fragment Substances 0.000 description 15
- 229920001184 polypeptide Polymers 0.000 description 15
- 102000004196 processed proteins & peptides Human genes 0.000 description 15
- 210000003527 eukaryotic cell Anatomy 0.000 description 13
- 238000000338 in vitro Methods 0.000 description 13
- 238000010453 CRISPR/Cas method Methods 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 11
- -1 grnas Proteins 0.000 description 11
- 238000009396 hybridization Methods 0.000 description 11
- 238000001890 transfection Methods 0.000 description 11
- 230000035772 mutation Effects 0.000 description 10
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 9
- 108020004682 Single-Stranded DNA Proteins 0.000 description 9
- 230000004048 modification Effects 0.000 description 9
- 238000012986 modification Methods 0.000 description 9
- 244000241257 Cucumis melo Species 0.000 description 8
- 241000700605 Viruses Species 0.000 description 8
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 8
- 230000008827 biological function Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 210000001236 prokaryotic cell Anatomy 0.000 description 8
- 108020004705 Codon Proteins 0.000 description 7
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 7
- 240000008042 Zea mays Species 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 241000894007 species Species 0.000 description 7
- 210000004102 animal cell Anatomy 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 125000006850 spacer group Chemical group 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 5
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 208000035475 disorder Diseases 0.000 description 5
- 235000013399 edible fruits Nutrition 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 235000009973 maize Nutrition 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 239000013603 viral vector Substances 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 4
- 244000291564 Allium cepa Species 0.000 description 4
- 108091033409 CRISPR Proteins 0.000 description 4
- 230000007018 DNA scission Effects 0.000 description 4
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 4
- 240000007594 Oryza sativa Species 0.000 description 4
- 235000007164 Oryza sativa Nutrition 0.000 description 4
- 230000007022 RNA scission Effects 0.000 description 4
- 240000003829 Sorghum propinquum Species 0.000 description 4
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 4
- 235000021307 Triticum Nutrition 0.000 description 4
- 241000209140 Triticum Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 238000004422 calculation algorithm Methods 0.000 description 4
- 239000013599 cloning vector Substances 0.000 description 4
- 238000007405 data analysis Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 239000012636 effector Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 230000002746 orthostatic effect Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 238000010791 quenching Methods 0.000 description 4
- 230000000171 quenching effect Effects 0.000 description 4
- 235000009566 rice Nutrition 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 241000701161 unidentified adenovirus Species 0.000 description 4
- 235000013311 vegetables Nutrition 0.000 description 4
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 3
- 240000007124 Brassica oleracea Species 0.000 description 3
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 3
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 3
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 3
- 244000221633 Brassica rapa subsp chinensis Species 0.000 description 3
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 3
- 235000007542 Cichorium intybus Nutrition 0.000 description 3
- 244000298479 Cichorium intybus Species 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- 241000195493 Cryptophyta Species 0.000 description 3
- 241000219130 Cucurbita pepo subsp. pepo Species 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 108010042407 Endonucleases Proteins 0.000 description 3
- 240000003183 Manihot esculenta Species 0.000 description 3
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 3
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 3
- 235000002597 Solanum melongena Nutrition 0.000 description 3
- 244000061458 Solanum melongena Species 0.000 description 3
- 230000002411 adverse Effects 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000001212 derivatisation Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000005714 functional activity Effects 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 210000005260 human cell Anatomy 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 238000000099 in vitro assay Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 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 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 238000001638 lipofection Methods 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 230000030648 nucleus localization Effects 0.000 description 3
- 230000008488 polyadenylation Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 125000006853 reporter group Chemical group 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241001430294 unidentified retrovirus Species 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 2
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 2
- 244000105624 Arachis hypogaea Species 0.000 description 2
- 235000007319 Avena orientalis Nutrition 0.000 description 2
- 241000209763 Avena sativa Species 0.000 description 2
- 235000007558 Avena sp Nutrition 0.000 description 2
- 235000016068 Berberis vulgaris Nutrition 0.000 description 2
- 241000335053 Beta vulgaris Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000167854 Bourreria succulenta Species 0.000 description 2
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 description 2
- 235000017647 Brassica oleracea var italica Nutrition 0.000 description 2
- 240000003259 Brassica oleracea var. botrytis Species 0.000 description 2
- 244000304217 Brassica oleracea var. gongylodes Species 0.000 description 2
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 description 2
- 101100011365 Caenorhabditis elegans egl-13 gene Proteins 0.000 description 2
- 241000282465 Canis Species 0.000 description 2
- 240000007154 Coffea arabica Species 0.000 description 2
- 101100329224 Coprinopsis cinerea (strain Okayama-7 / 130 / ATCC MYA-4618 / FGSC 9003) cpf1 gene Proteins 0.000 description 2
- 241000711573 Coronaviridae Species 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 235000003954 Cucurbita pepo var melopepo Nutrition 0.000 description 2
- 244000019459 Cynara cardunculus Species 0.000 description 2
- 235000019106 Cynara scolymus Nutrition 0.000 description 2
- 241000702421 Dependoparvovirus Species 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 241000219146 Gossypium Species 0.000 description 2
- 208000009889 Herpes Simplex Diseases 0.000 description 2
- 235000003228 Lactuca sativa Nutrition 0.000 description 2
- 240000008415 Lactuca sativa Species 0.000 description 2
- 101710128836 Large T antigen Proteins 0.000 description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 2
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 2
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 2
- 244000061176 Nicotiana tabacum Species 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 2
- 244000046052 Phaseolus vulgaris Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 240000003768 Solanum lycopersicum Species 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 235000009470 Theobroma cacao Nutrition 0.000 description 2
- 244000299461 Theobroma cacao Species 0.000 description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 2
- 244000078534 Vaccinium myrtillus Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 235000016520 artichoke thistle Nutrition 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 101150059443 cas12a gene Proteins 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 235000019693 cherries Nutrition 0.000 description 2
- 235000016213 coffee Nutrition 0.000 description 2
- 235000013353 coffee beverage Nutrition 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 210000001808 exosome Anatomy 0.000 description 2
- 235000021384 green leafy vegetables Nutrition 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 235000014571 nuts Nutrition 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 2
- 230000004952 protein activity Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 230000037426 transcriptional repression Effects 0.000 description 2
- 101150059931 tus gene Proteins 0.000 description 2
- 239000000277 virosome Substances 0.000 description 2
- AUXMWYRZQPIXCC-KNIFDHDWSA-N (2s)-2-amino-4-methylpentanoic acid;(2s)-2-aminopropanoic acid Chemical compound C[C@H](N)C(O)=O.CC(C)C[C@H](N)C(O)=O AUXMWYRZQPIXCC-KNIFDHDWSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 description 1
- 208000035657 Abasia Diseases 0.000 description 1
- 240000004507 Abelmoschus esculentus Species 0.000 description 1
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 235000009436 Actinidia deliciosa Nutrition 0.000 description 1
- 244000298697 Actinidia deliciosa Species 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- WQVFQXXBNHHPLX-ZKWXMUAHSA-N Ala-Ala-His Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O WQVFQXXBNHHPLX-ZKWXMUAHSA-N 0.000 description 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- 235000005254 Allium ampeloprasum Nutrition 0.000 description 1
- 240000006108 Allium ampeloprasum Species 0.000 description 1
- 235000010167 Allium cepa var aggregatum Nutrition 0.000 description 1
- 240000002234 Allium sativum Species 0.000 description 1
- 241000143060 Americamysis bahia Species 0.000 description 1
- 244000144725 Amygdalus communis Species 0.000 description 1
- 244000099147 Ananas comosus Species 0.000 description 1
- 235000007119 Ananas comosus Nutrition 0.000 description 1
- 244000105975 Antidesma platyphyllum Species 0.000 description 1
- 235000003276 Apios tuberosa Nutrition 0.000 description 1
- 240000007087 Apium graveolens Species 0.000 description 1
- 235000015849 Apium graveolens Dulce Group Nutrition 0.000 description 1
- 235000010591 Appio Nutrition 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000010744 Arachis villosulicarpa Nutrition 0.000 description 1
- PTVGLOCPAVYPFG-CIUDSAMLSA-N Arg-Gln-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O PTVGLOCPAVYPFG-CIUDSAMLSA-N 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- PTNFNTOBUDWHNZ-GUBZILKMSA-N Asn-Arg-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O PTNFNTOBUDWHNZ-GUBZILKMSA-N 0.000 description 1
- LJUOLNXOWSWGKF-ACZMJKKPSA-N Asn-Asn-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N LJUOLNXOWSWGKF-ACZMJKKPSA-N 0.000 description 1
- KHCNTVRVAYCPQE-CIUDSAMLSA-N Asn-Lys-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O KHCNTVRVAYCPQE-CIUDSAMLSA-N 0.000 description 1
- FANQWNCPNFEPGZ-WHFBIAKZSA-N Asp-Asp-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O FANQWNCPNFEPGZ-WHFBIAKZSA-N 0.000 description 1
- 244000003416 Asparagus officinalis Species 0.000 description 1
- 235000005340 Asparagus officinalis Nutrition 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 235000000832 Ayote Nutrition 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000093727 Berzelia alopecuroides Species 0.000 description 1
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- 241000237519 Bivalvia Species 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 235000003351 Brassica cretica Nutrition 0.000 description 1
- 235000011293 Brassica napus Nutrition 0.000 description 1
- 235000001171 Brassica oleracea var gongylodes Nutrition 0.000 description 1
- 240000008100 Brassica rapa Species 0.000 description 1
- 235000000540 Brassica rapa subsp rapa Nutrition 0.000 description 1
- 235000003343 Brassica rupestris Nutrition 0.000 description 1
- 235000004936 Bromus mango Nutrition 0.000 description 1
- 208000025721 COVID-19 Diseases 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 241000219357 Cactaceae Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 235000012766 Cannabis sativa ssp. sativa var. sativa Nutrition 0.000 description 1
- 235000012765 Cannabis sativa ssp. sativa var. spontanea Nutrition 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 235000009467 Carica papaya Nutrition 0.000 description 1
- 240000006432 Carica papaya Species 0.000 description 1
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 1
- 244000020518 Carthamus tinctorius Species 0.000 description 1
- 235000009025 Carya illinoensis Nutrition 0.000 description 1
- 244000068645 Carya illinoensis Species 0.000 description 1
- 235000014036 Castanea Nutrition 0.000 description 1
- 241001070941 Castanea Species 0.000 description 1
- 235000003301 Ceiba pentandra Nutrition 0.000 description 1
- 244000146553 Ceiba pentandra Species 0.000 description 1
- 235000010523 Cicer arietinum Nutrition 0.000 description 1
- 244000045195 Cicer arietinum Species 0.000 description 1
- 244000223760 Cinnamomum zeylanicum Species 0.000 description 1
- 244000241235 Citrullus lanatus Species 0.000 description 1
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 235000013162 Cocos nucifera Nutrition 0.000 description 1
- 244000060011 Cocos nucifera Species 0.000 description 1
- 241000218631 Coniferophyta Species 0.000 description 1
- 240000000491 Corchorus aestuans Species 0.000 description 1
- 235000011777 Corchorus aestuans Nutrition 0.000 description 1
- 235000010862 Corchorus capsularis Nutrition 0.000 description 1
- 241000723382 Corylus Species 0.000 description 1
- 235000007466 Corylus avellana Nutrition 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 235000009847 Cucumis melo var cantalupensis Nutrition 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- 235000009854 Cucurbita moschata Nutrition 0.000 description 1
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 description 1
- 235000017788 Cydonia oblonga Nutrition 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 235000011511 Diospyros Nutrition 0.000 description 1
- 241000723267 Diospyros Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 206010017472 Fumbling Diseases 0.000 description 1
- 102100039556 Galectin-4 Human genes 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- NUSWUSKZRCGFEX-FXQIFTODSA-N Glu-Glu-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CS)C(O)=O NUSWUSKZRCGFEX-FXQIFTODSA-N 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 108010014594 Heterogeneous Nuclear Ribonucleoprotein A1 Proteins 0.000 description 1
- 102000017013 Heterogeneous Nuclear Ribonucleoprotein A1 Human genes 0.000 description 1
- 244000043261 Hevea brasiliensis Species 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000608765 Homo sapiens Galectin-4 Proteins 0.000 description 1
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 206010020460 Human T-cell lymphotropic virus type I infection Diseases 0.000 description 1
- 241000714260 Human T-lymphotropic virus 1 Species 0.000 description 1
- 241000711467 Human coronavirus 229E Species 0.000 description 1
- 241001109669 Human coronavirus HKU1 Species 0.000 description 1
- 241000482741 Human coronavirus NL63 Species 0.000 description 1
- 241001428935 Human coronavirus OC43 Species 0.000 description 1
- 235000008694 Humulus lupulus Nutrition 0.000 description 1
- 244000025221 Humulus lupulus Species 0.000 description 1
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 1
- 235000010702 Insulata Nutrition 0.000 description 1
- 244000165077 Insulata Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 241000758791 Juglandaceae Species 0.000 description 1
- 235000015802 Lactuca sativa var crispa Nutrition 0.000 description 1
- 240000004201 Lactuca sativa var. crispa Species 0.000 description 1
- 241000283953 Lagomorpha Species 0.000 description 1
- 240000004322 Lens culinaris Species 0.000 description 1
- 235000014647 Lens culinaris subsp culinaris Nutrition 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 235000004431 Linum usitatissimum Nutrition 0.000 description 1
- 240000006240 Linum usitatissimum Species 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 241000218922 Magnoliophyta Species 0.000 description 1
- 244000070406 Malus silvestris Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 244000307609 Mangifera caesia Species 0.000 description 1
- 235000014826 Mangifera indica Nutrition 0.000 description 1
- 240000007228 Mangifera indica Species 0.000 description 1
- 235000010804 Maranta arundinacea Nutrition 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 240000005561 Musa balbisiana Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 244000183278 Nephelium litchi Species 0.000 description 1
- 241000219925 Oenothera Species 0.000 description 1
- 235000004496 Oenothera biennis Nutrition 0.000 description 1
- 241000207836 Olea <angiosperm> Species 0.000 description 1
- 240000001090 Papaver somniferum Species 0.000 description 1
- 235000008753 Papaver somniferum Nutrition 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 240000004370 Pastinaca sativa Species 0.000 description 1
- 235000017769 Pastinaca sativa subsp sativa Nutrition 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 235000008673 Persea americana Nutrition 0.000 description 1
- 244000025272 Persea americana Species 0.000 description 1
- 244000062780 Petroselinum sativum Species 0.000 description 1
- WEMYTDDMDBLPMI-DKIMLUQUSA-N Phe-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N WEMYTDDMDBLPMI-DKIMLUQUSA-N 0.000 description 1
- YTILBRIUASDGBL-BZSNNMDCSA-N Phe-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 YTILBRIUASDGBL-BZSNNMDCSA-N 0.000 description 1
- KIQUCMUULDXTAZ-HJOGWXRNSA-N Phe-Tyr-Tyr Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O KIQUCMUULDXTAZ-HJOGWXRNSA-N 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 240000003889 Piper guineense Species 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- 240000006711 Pistacia vera Species 0.000 description 1
- 235000010582 Pisum sativum Nutrition 0.000 description 1
- 240000004713 Pisum sativum Species 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 235000009827 Prunus armeniaca Nutrition 0.000 description 1
- 244000018633 Prunus armeniaca Species 0.000 description 1
- 244000141353 Prunus domestica Species 0.000 description 1
- 240000005809 Prunus persica Species 0.000 description 1
- 235000006029 Prunus persica var nucipersica Nutrition 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 244000017714 Prunus persica var. nucipersica Species 0.000 description 1
- 235000014360 Punica granatum Nutrition 0.000 description 1
- 244000294611 Punica granatum Species 0.000 description 1
- 241000220324 Pyrus Species 0.000 description 1
- 244000088415 Raphanus sativus Species 0.000 description 1
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 235000011552 Rhamnus crocea Nutrition 0.000 description 1
- 235000001537 Ribes X gardonianum Nutrition 0.000 description 1
- 235000001535 Ribes X utile Nutrition 0.000 description 1
- 235000016919 Ribes petraeum Nutrition 0.000 description 1
- 244000281247 Ribes rubrum Species 0.000 description 1
- 235000002355 Ribes spicatum Nutrition 0.000 description 1
- 240000000528 Ricinus communis Species 0.000 description 1
- 235000004443 Ricinus communis Nutrition 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 240000007651 Rubus glaucus Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 241000209056 Secale Species 0.000 description 1
- 235000007238 Secale cereale Nutrition 0.000 description 1
- QMCDMHWAKMUGJE-IHRRRGAJSA-N Ser-Phe-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O QMCDMHWAKMUGJE-IHRRRGAJSA-N 0.000 description 1
- FZXOPYUEQGDGMS-ACZMJKKPSA-N Ser-Ser-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O FZXOPYUEQGDGMS-ACZMJKKPSA-N 0.000 description 1
- DKGRNFUXVTYRAS-UBHSHLNASA-N Ser-Ser-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O DKGRNFUXVTYRAS-UBHSHLNASA-N 0.000 description 1
- 235000003434 Sesamum indicum Nutrition 0.000 description 1
- 244000040738 Sesamum orientale Species 0.000 description 1
- 244000044822 Simmondsia californica Species 0.000 description 1
- 235000004433 Simmondsia californica Nutrition 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000009337 Spinacia oleracea Nutrition 0.000 description 1
- 244000300264 Spinacia oleracea Species 0.000 description 1
- 235000009184 Spondias indica Nutrition 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- 244000145580 Thalia geniculata Species 0.000 description 1
- 235000012419 Thalia geniculata Nutrition 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 1
- 241000219793 Trifolium Species 0.000 description 1
- ARJASMXQBRNAGI-YESZJQIVSA-N Tyr-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N ARJASMXQBRNAGI-YESZJQIVSA-N 0.000 description 1
- 235000003095 Vaccinium corymbosum Nutrition 0.000 description 1
- 235000017537 Vaccinium myrtillus Nutrition 0.000 description 1
- 241000219094 Vitaceae Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 108091093126 WHP Posttrascriptional Response Element Proteins 0.000 description 1
- 241000482268 Zea mays subsp. mays Species 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 235000020224 almond Nutrition 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000021016 apples Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 235000021015 bananas Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 description 1
- 235000021029 blackberry Nutrition 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 235000021014 blueberries Nutrition 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 235000009120 camo Nutrition 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000005607 chanvre indien Nutrition 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 235000017803 cinnamon Nutrition 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 235000020639 clam Nutrition 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000002856 computational phylogenetic analysis Methods 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 244000038559 crop plants Species 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000002050 diffraction method Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 238000000835 electrochemical detection Methods 0.000 description 1
- 238000002003 electron diffraction Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 241001233957 eudicotyledons Species 0.000 description 1
- 244000037666 field crops Species 0.000 description 1
- 238000002875 fluorescence polarization Methods 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000004459 forage Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 235000004611 garlic Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 235000021021 grapes Nutrition 0.000 description 1
- 235000009424 haa Nutrition 0.000 description 1
- 239000011487 hemp Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002923 metal particle Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 235000010460 mustard Nutrition 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000006780 non-homologous end joining Effects 0.000 description 1
- 230000025308 nuclear transport Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 235000021017 pears Nutrition 0.000 description 1
- 235000011197 perejil Nutrition 0.000 description 1
- 238000005222 photoaffinity labeling Methods 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 235000020233 pistachio Nutrition 0.000 description 1
- 235000021018 plums Nutrition 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 235000013594 poultry meat Nutrition 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000002818 protein evolution Methods 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 235000015136 pumpkin Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 235000021013 raspberries Nutrition 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 230000003007 single stranded DNA break Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 235000021012 strawberries Nutrition 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of nucleic acid editing, in particular to the technical field of regularly clustered interval short palindromic repeat (CRISPR). Specifically, the invention provides a novel Cas enzyme, which has low homology with the reported Cas enzyme, can realize nuclease activity in cells and outside cells, and has wide application prospect.
Description
Technical Field
The invention relates to the field of gene editing, in particular to the technical field of regular clustered interval short palindromic repeat (CRISPR). In particular, the invention relates to a novel CRISPR enzyme (alternatively referred to as CRISPR protein, cas effect protein, cas enzyme or Cas protein), fusion proteins comprising such proteins, and nucleic acid molecules encoding them. The invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing) comprising the Cas protein or fusion protein of the invention, or nucleic acid molecules encoding the same.
Background
CRISPR/Cas technology is a widely used gene editing technology that uses RNA-guided specific binding of target sequences on the genome and cleavage of DNA to create double strand breaks, site-directed gene editing using biological non-homologous end joining or homologous recombination.
The CRISPR/Cas9 system is the most commonly used type II CRISPR system that recognizes the PAM motif of 3' -NGG and blunt-ends the target sequence. The CRISPR/Cas Type V system is a newly discovered class of CRISPR systems that have a 5' -TTN motif that performs cohesive end cleavage of a target sequence, e.g., cpf1, C2C1, casX, casY. However, the different CRISPR/Cas currently in existence each have different advantages and disadvantages. For example, cas9, C2C1 and CasX each require two RNAs for guide RNAs, whereas Cpf1 requires only one guide RNA and can be used for multiplex gene editing. CasX has a size of 980 amino acids, whereas common Cas9, C2C1, casY and Cpf1 are typically around 1300 amino acids in size. In addition, PAM sequences of Cas9, cpf1, casX, casY are all relatively complex and diverse, while C2C1 recognizes the stringent 5' -TTN, so its target site is easily predicted compared to other systems, thereby reducing potential off-target effects.
In summary, given that the currently available CRISPR/Cas systems are limited by several drawbacks, the development of a more robust new CRISPR/Cas system with versatile good performance is of great importance for the development of biotechnology.
Disclosure of Invention
The inventors of the present application have unexpectedly discovered a novel endonuclease (Cas enzyme) through a number of experiments and repeated fumbling. Based on this finding, the present inventors developed a new CRISPR/Cas system and a gene editing method and a nucleic acid detection method based on the same.
Cas effector proteins
In one aspect, the present invention provides a Cas protein, which is an effector protein in a CRISPR/Cas system, herein referred to as Cas-sf0009 (shown in SEQ ID No. 1), cas-sf1782 (shown in SEQ ID No. 4), cas-sf2431 (shown in SEQ ID No. 7); the phylogenetic tree analysis shows that the Cas proteins described above belong to Cas proteins of the Cas12j family.
In one embodiment, the Cas protein amino acid sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity as compared to SEQ ID No.1, and substantially retains the biological function of the sequence from which it is derived. Preferably, the Cas protein is derived from the same species as Cas-sf 0009.
In one embodiment, the Cas protein amino acid sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity as compared to SEQ ID No.4, and substantially retains the biological function of the sequence from which it is derived. Preferably, the Cas protein is derived from the same species as Cas-sf 1782.
In one embodiment, the Cas protein amino acid sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity as compared to SEQ ID No.7, and substantially retains the biological function of the sequence from which it is derived. Preferably, the Cas protein is derived from the same species as Cas-sf 2431.
In one embodiment, the Cas protein amino acid sequence has a sequence of substitution, deletion, or addition of one or more amino acids as compared to SEQ ID No. 1; and substantially retains the biological function of the sequence from which it originates; the substitution, deletion or addition of one or more amino acids includes substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Preferably, the Cas protein is derived from the same species as Cas-sf 0009.
In one embodiment, the Cas protein amino acid sequence has a sequence of substitution, deletion, or addition of one or more amino acids as compared to SEQ ID No. 4; and substantially retains the biological function of the sequence from which it originates; the substitution, deletion or addition of one or more amino acids includes substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Preferably, the Cas protein is derived from the same species as Cas-sf 1782.
In one embodiment, the Cas protein amino acid sequence has a sequence of substitution, deletion, or addition of one or more amino acids as compared to SEQ ID No. 7; and substantially retains the biological function of the sequence from which it originates; the substitution, deletion or addition of one or more amino acids includes substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Preferably, the Cas protein is derived from the same species as Cas-sf 2431.
It will be apparent to those skilled in the art that the structure of a protein may be altered without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions may be introduced into the amino acid sequence of the protein without adversely affecting the activity and/or three-dimensional structure of the protein molecule. Examples and embodiments of conservative amino acid substitutions are apparent to those skilled in the art. In particular, the amino acid residue may be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., with a nonpolar amino acid residue, with a polar uncharged amino acid residue, with a basic amino acid residue, with an acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions of one amino acid by another belonging to the same group are within the scope of the invention as long as the substitution does not result in inactivation of the biological activity of the protein. Thus, the proteins of the invention may comprise one or more conservative substitutions in the amino acid sequence, which are preferably made according to table 1. In addition, proteins that also contain one or more other non-conservative substitutions are also contemplated by the present invention, provided that the non-conservative substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.
Conservative amino acid substitutions may be made at one or more predicted nonessential amino acid residues. "nonessential" amino acid residues are amino acid residues that can be altered (deleted, substituted or substituted) without altering the biological activity, whereas "essential" amino acid residues are required for the biological activity. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acid substitutions may be made in non-conserved regions of the Cas proteins described above. In general, such substitutions are not made to conserved amino acid residues, or amino acid residues that are within a conserved motif, where such residues are required for protein activity. However, it will be appreciated by those skilled in the art that functional variants may have fewer conservative or non-conservative changes in the conserved regions.
TABLE 1
Initial residues | Representative substitution | Preferred substitution |
Ala(A) | Val;Leu;Ile | Val |
Arg(R) | Lys;Gln;Asn | Lys |
Asn(N) | Gln;His;Lys;Arg | Gln |
Asp(D) | Glu | Glu |
Cys(C) | Ser | Ser |
Gln(Q) | Asn | Asn |
Glu(E) | Asp | Asp |
Gly(G) | Pro;Ala | Ala |
His(H) | Asn;Gln;Lys;Arg | Arg |
Ile(I) | Leu;Val;Met;Ala;Phe | Leu |
Leu(L) | Ile;Val;Met;Ala;Phe | Ile |
Lys(K) | Arg;Gln;Asn | Arg |
Met(M) | Leu;Phe;Ile | Leu |
Phe(F) | Leu;Val;Ile;Ala;Tyr | Leu |
Pro(P) | Ala | Ala |
Ser(S) | Thr | Thr |
Thr(T) | Ser | Ser |
Trp(W) | Tyr;Phe | Tyr |
Tyr(Y) | Trp;Phe;Thr;Ser | Phe |
Val(V) | Ile;Leu;Met;Phe;Ala | Leu |
It is well known in the art that one or more amino acid residues may be altered (substituted, deleted, truncated or inserted) from the N-and/or C-terminus of a protein while still retaining its functional activity. Thus, proteins that have one or more amino acid residues altered from the N and/or C terminus of the Cas protein while retaining their desired functional activity are also within the scope of the invention. These changes may include changes introduced by modern molecular methods such as PCR, including PCR amplification that alters or extends the protein coding sequence by including an amino acid coding sequence in the oligonucleotides used in the PCR amplification.
It will be appreciated that proteins may be altered in a variety of ways, including amino acid substitutions, deletions, truncations and insertions, and that methods for such manipulation are generally known in the art. For example, amino acid sequence variants of the above proteins can be prepared by mutation of DNA. Single or multiple amino acid substitutions, deletions and/or insertions may also be made by other forms of mutagenesis and/or by directed evolution, for example, using known mutagenesis, recombination and/or shuffling (shuffleling) methods, in combination with associated screening methods.
Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the invention may occur (e.g., naturally occurring mutations) or be generated (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site or other functional domain of the protein, the nature of the polypeptide may be altered, but the polypeptide may retain its activity. Smaller effects can be expected if mutations are present that are not close to the catalytic domain, active site or other functional domain.
The skilled artisan can identify the essential amino acids of the Cas protein of the invention according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of bioinformatics. The catalytic, active or other functional domains of a protein can also be determined by physical analysis of the structure, such as by the following techniques: such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations in the amino acids at putative key sites.
In one embodiment, the Cas protein contains the amino acid sequence set forth in SEQ ID No.1, SEQ ID No.4, or SEQ ID No. 7.
In one embodiment, the Cas protein is the amino acid sequence shown as SEQ ID No.1, SEQ ID No.4, or SEQ ID No. 7.
In one embodiment, the Cas protein is a derivatized protein having the same biological function as a protein having the sequence shown in SEQ ID No.1, SEQ ID No.4, or SEQ ID No. 7.
Such biological functions include, but are not limited to, activity of binding to a guide RNA, endonuclease activity, activity of binding to and cleaving at a specific site of a target sequence under the guidance of a guide RNA, including, but not limited to, cis cleavage activity and Trans cleavage activity.
The invention also provides a fusion protein comprising a Cas protein as described above and other modifying moieties.
In one embodiment, the modifying moiety is selected from the group consisting of an additional protein or polypeptide, a detectable label, or any combination thereof.
In one embodiment, the modifying moiety is selected from the group consisting of an epitope tag, a reporter gene sequence, a Nuclear Localization Signal (NLS) sequence, a targeting moiety, a transcriptional activation domain (e.g., VP 64), a transcriptional repression domain (e.g., KRAB domain or SID domain), a nuclease domain (e.g., fok 1), and a domain having an activity selected from the group consisting of: nucleotide deaminase, methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity and nucleic acid binding activity; and any combination thereof. The NLS sequences are well known to those skilled in the art, examples of which include, but are not limited to, the SV40 large T antigen, EGL-13, c-Myc, and TUS proteins.
In one embodiment, the NLS sequence is located at, near, or near the terminus (e.g., N-terminus, C-terminus, or both) of the Cas protein of the invention.
Such epitope tags (tags) are well known to those skilled in the art, including but not limited to His, V5, FLAG, HA, myc, VSV-G, trx, etc., and other suitable epitope tags (e.g., purification, detection, or labeling) may be selected by those skilled in the art.
Such reporter sequences are well known to those skilled in the art, examples of which include, but are not limited to GST, HRP, CAT, GFP, hcRed, dsRed, CFP, YFP, BFP, etc.
In one embodiment, the fusion proteins of the invention comprise a domain capable of binding to a DNA molecule or an intracellular molecule, such as Maltose Binding Protein (MBP), the DNA binding domain of Lex a (DBD), the DBD of GAL4, and the like.
In one embodiment, the fusion proteins of the invention comprise a detectable label, such as a fluorescent dye, e.g., FITC or DAPI.
In one embodiment, the Cas protein of the invention is coupled, conjugated or fused to the modifying moiety, optionally through a linker.
In one embodiment, the modification is directly linked to the N-terminus or C-terminus of the Cas protein of the invention.
In one embodiment, the modifying moiety is linked to the N-terminus or C-terminus of the Cas protein of the invention by a linker. Such linkers are well known in the art, examples of which include, but are not limited to, linkers comprising one or more (e.g., 1, 2, 3, 4, or 5) amino acids (e.g., glu or Ser) or amino acid derivatives (e.g., ahx, β -Ala, GABA, or Ava), or PEG, etc.
The Cas protein, protein derivative or fusion protein of the present invention is not limited by the manner of its production, and for example, it may be produced by genetic engineering methods (recombinant techniques) or by chemical synthesis methods.
Nucleic acid of Cas protein
In another aspect, the invention provides an isolated polynucleotide comprising:
(a) Polynucleotide sequences encoding Cas proteins or fusion proteins of the invention;
or,
(b) Polynucleotides of sequence SEQ ID No.2, 3, 5, 6, 8 or 9;
or,
(c) A polynucleotide complementary to the polynucleotide of any one of (a) - (b).
In one embodiment, the nucleotide sequence set forth in any one of (a) - (c) is codon optimized for expression in a prokaryotic cell. In one embodiment, the nucleotide sequence set forth in any one of (a) - (c) is codon optimized for expression in a eukaryotic cell.
In one embodiment, the cell is an animal cell, e.g., a mammalian cell.
In one embodiment, the cell is a human cell.
In one embodiment, the cell is a plant cell, such as a cell of a cultivated plant (e.g., cassava, maize, sorghum, wheat, or rice), algae, tree, or vegetable.
In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.
Direct repeat (directread) sequence
In another aspect, the invention provides an engineered homodromous repeat sequence that forms a complex with the Cas protein described above.
The orthostatic repeat sequence is linked to a guide sequence capable of hybridizing to a target sequence to form a guide RNA (guide RNA or gRNA).
Hybridization of the target sequence to the gRNA, representing at least 70%,75%,80%,85%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or 100% identity of the target sequence to the nucleic acid sequence of the gRNA, such that hybridization can form a complex; or at least 12, 15, 16, 17, 18, 19, 20, 21, 22, or more bases of the nucleic acid sequence representing the target sequence and the gRNA may be complementarily paired to form a complex.
In some embodiments, the orthostatic sequence has at least 90% sequence identity to any one of SEQ ID Nos. 10-12. In some embodiments, the orthostatic sequence has a substitution, deletion, or addition of one or more bases (e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases) as compared to the sequence set forth in any one of SEQ ID nos. 10-12.
In some embodiments, the orthostatic repeat sequence is as set forth in any one of SEQ ID Nos. 10-12.
Guide RNA (gRNA)
In another aspect, the invention provides a gRNA comprising a first segment and a second segment; the first segment is also known as a "framework region", "protein binding segment", "protein binding sequence", or "Direct Repeat (Direct Repeat) sequence"; the second segment is also referred to as a "targeting sequence of a targeting nucleic acid" or a "targeting segment of a targeting nucleic acid", or a "targeting sequence of a targeting nucleic acid".
The first segment of the gRNA is capable of interacting with the Cas protein of the invention, thereby forming a complex of Cas protein and gRNA.
In a preferred embodiment, the first segment is a homeotropic repeat as described above.
The targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid of the invention comprises a nucleotide sequence complementary to a sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid of the invention interacts with the target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing). Thus, the targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid may be altered, or may be modified to hybridize to any desired sequence within the target nucleic acid. The nucleic acid is selected from DNA or RNA.
The targeting sequence of the targeting nucleic acid or the percentage of complementarity between the targeting segment of the targeting nucleic acid and the target sequence of the target nucleic acid can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).
The "framework region", "protein binding segment", "protein binding sequence", or "cognate repeat" of the gRNA of the invention can interact with a CRISPR protein (or Cas protein). The gRNA of the invention directs its interacting Cas protein to a specific nucleotide sequence within the target nucleic acid through the action of the targeting sequence of the targeting nucleic acid.
Preferably, the guide RNA comprises a first segment and a second segment in the 5 'to 3' direction.
In the context of the present invention, the second segment is also understood as a guide sequence which hybridizes to the target sequence.
The gRNA of the invention is capable of forming a complex with the Cas protein.
The gRNA of the Cas-sf0009 protein of the invention comprises a guide sequence that hybridizes to a target nucleic acid, wherein the target nucleic acid comprises a sequence located 3' of a Protospacer Adjacent Motif (PAM); the PAM sequence is 5'-NTT-3',5'-ATD-3',5'-TTN-3' or 5'-STT-3', wherein N=A/T/C/G, S=C/G, D=A/G/T.
The gRNA of the Cas-sf1782 protein of the invention comprises a guide sequence that hybridizes to a target nucleic acid, wherein the target nucleic acid comprises a sequence located 3' of a Protospacer Adjacent Motif (PAM); the PAM sequence is 5'-NTN-3',5'-NTT-3', 5'-TTC-3' or 5'-CAA-3', wherein N=A/T/C/G.
The gRNA of the Cas-sf2431 protein of the invention comprises a guide sequence that hybridizes to a target nucleic acid, wherein the target nucleic acid comprises a sequence located 3' of a Protospacer Adjacent Motif (PAM); the PAM sequence is 5' -TTC-3.
Carrier body
The invention also provides a vector comprising a Cas protein, an isolated nucleic acid molecule, or a polynucleotide as described above; preferably, it further comprises a regulatory element operably linked thereto.
In one embodiment, the regulatory element is selected from one or more of the following group: enhancers, transposons, promoters, terminators, leader sequences, polyadenylation sequences, and marker genes.
In one embodiment, the vector comprises a cloning vector, an expression vector, a shuttle vector, an integration vector.
In some embodiments, the vectors included in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), but may also be of the plasmid, viral, cosmid, phage, etc. type, which are well known to those skilled in the art.
CRISPR system
The present invention provides an engineered non-naturally occurring vector system, or CRISPR-Cas system, comprising a Cas protein or a nucleic acid sequence encoding the Cas protein and a nucleic acid encoding one or more guide RNAs.
In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more guide RNAs are synthetic.
In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more guide RNAs do not co-occur naturally.
The one or more guide RNAs target one or more target sequences in the cell. The one or more target sequences hybridize to a genomic locus of a DNA molecule encoding one or more gene products and the Cas protein is directed to the genomic locus of the DNA molecule of the one or more gene products, and the Cas protein, upon reaching the target sequence position, modifies, edits or cleaves the target sequence, whereby expression of the one or more gene products is altered or modified.
The cells of the invention include one or more of animals, plants or microorganisms.
In some embodiments, the Cas protein is codon optimized for expression in a cell.
In some embodiments, the Cas protein cleaves one or both strands at the target sequence position.
In some embodiments, the Cas protein cleaves the complementary strand and/or the non-complementary strand of the target nucleic acid under the mediation of the gRNA.
In the present invention, the gRNA directs Cas protein recognition and binding on the complementary strand, and the non-complementary strand is a nucleic acid strand paired with the complementary strand. The PAM sequence is located on a non-complementary strand that contains a PAM complementary sequence paired with the PAM sequence described above.
In one embodiment, the cleavage site of the complementary strand of Cas-sf0009 to the target sequence is between 23nt and 24nt of the 5 'end of the PAM complementary sequence, the cleavage site of the non-complementary strand of Cas-sf0009 to the target sequence is between 20nt and 21nt of the 3' end of the PAM sequence, and the gRNA directs Cas-sf0009 protein to recognize and bind to the complementary strand, which is the DNA strand paired with the complementary strand.
The invention also provides an engineered non-naturally occurring carrier system that can include one or more carriers comprising:
a) A first regulatory element operably linked to the gRNA,
b) A second regulatory element operably linked to the Cas protein;
wherein components (a) and (b) are on the same or different supports of the system.
The first and second regulatory elements include promoters (e.g., constitutive promoters or inducible promoters), enhancers (e.g., 35S promoter or 35S enhanced promoter), internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcriptional termination signals, such as polyadenylation signals and poly U sequences).
In some embodiments, the vectors in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), but may also be of the plasmid, viral, cosmid, phage, etc. type, which are well known to those skilled in the art.
In some embodiments, the systems provided herein are in a delivery system. In some embodiments, the delivery system is a nanoparticle, liposome, exosome, microvesicle, or gene gun.
In one embodiment, the target sequence is a DNA or RNA sequence from a prokaryotic or eukaryotic cell. In one embodiment, the target sequence is a non-naturally occurring DNA or RNA sequence.
In one embodiment, the target sequence is present in a cell. In one embodiment, the target sequence is present in the nucleus or in the cytoplasm (e.g., organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.
In one embodiment, the Cas protein has one or more NLS sequences attached. In one embodiment, the fusion protein comprises one or more NLS sequences. In one embodiment, the NLS sequence is linked to the N-terminus or C-terminus of the protein. In one embodiment, the NLS sequence is fused to the N-terminus or C-terminus of the protein.
In another aspect, the invention relates to an engineered CRISPR system comprising the Cas protein described above and one or more guide RNAs, wherein the guide RNAs comprise a homodromous repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid, the Cas protein is capable of binding to the guide RNAs and targeting a target nucleic acid sequence complementary to the spacer sequence.
In one embodiment, the Cas enzyme is Cas-sf0009, the target nucleic acid is DNA (preferably, double-stranded DNA), the target nucleic acid is located adjacent to the 3' end of a motif (PAM) from a protospacer, and the PAM has a sequence shown as 5' -NTT-3',5' -ATD-3',5' -TTN-3', or 5' -STT-3', where n=a/T/C/G, s=c/G, d=a/G/T.
In one embodiment, the Cas enzyme is Cas-sf1782, the target nucleic acid is DNA (preferably, double-stranded DNA), the target nucleic acid is located at the 3' end of the Protospacer Adjacent Motif (PAM), and the PAM is 5' -NTN-3',5' -NTT-3',5' -TTC-3', or 5' -CAA-3', where n=a/T/C/G.
In one embodiment, the Cas enzyme is Cas-sf2431, the target nucleic acid is DNA (preferably, double-stranded DNA), the target nucleic acid is located 3' to the Protospacer Adjacent Motif (PAM), and the PAM is 5' -TTC-3'.
Protein-nucleic acid complexes/compositions
In another aspect, the invention provides a complex or composition comprising:
(i) A protein component selected from the group consisting of: the Cas protein, the derivatized protein, or the fusion protein described above, and any combination thereof; and
(ii) A nucleic acid component comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a homeotropic repeat capable of binding to the Cas protein of the invention.
The protein component and the nucleic acid component are bound to each other to form a complex.
In one embodiment, the nucleic acid component is a guide RNA in a CRISPR-Cas system.
In one embodiment, the complex or composition is non-naturally occurring or modified. In one embodiment, at least one component of the complex or composition is non-naturally occurring or modified. In one embodiment, the first component is non-naturally occurring or modified; and/or, the second component is non-naturally occurring or modified.
Activated CRISPR complexes
In another aspect, the present invention also provides an activated CRISPR complex comprising: (1) a protein component selected from the group consisting of: the Cas protein, the derivatized protein, or the fusion protein of the invention, and any combination thereof; (2) A gRNA comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a homeotropic repeat capable of binding to the Cas protein of the invention; and (3) a target sequence that binds to the gRNA. Preferably, the binding is binding to the target nucleic acid through a targeting sequence of the targeting nucleic acid on the gRNA.
The term "activated CRISPR complex", "activated complex" or "ternary complex" as used herein refers to a complex in a CRISPR system where Cas protein, gRNA bind to or are modified with a target nucleic acid.
The Cas proteins and grnas of the invention can form binary complexes that are activated upon binding to a nucleic acid substrate that is complementary to a spacer sequence in the gRNA (or, referred to as, a guide sequence that hybridizes to a target nucleic acid) to form an activated CRISPR complex. In some embodiments, the spacer sequence of the gRNA is perfectly matched to the target substrate. In other embodiments, the spacer sequence of the gRNA matches a portion (continuous or discontinuous) of the target substrate.
In preferred embodiments, the activated CRISPR complex may exhibit a sidebranch nuclease cleavage activity, which refers to the nonspecific cleavage activity or the nicking activity of the activated CRISPR complex on single stranded nucleic acids, also known in the art as trans cleavage activity.
Delivery and delivery compositions
Cas proteins, grnas, fusion proteins, nucleic acid molecules, vectors, systems, complexes, and compositions of the invention may be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nuclear transfection, microinjection, sonoporation, gene gun, calcium phosphate mediated transfection, cationic transfection, lipofection, dendritic transfection, heat shock transfection, nuclear transfection, magnetic transfection, lipofection, puncture transfection, optical transfection, reagent enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, virosomes, artificial virosomes, and the like.
Accordingly, in another aspect, the present invention provides a delivery composition comprising a delivery vehicle, and one or more selected from any of the following: cas proteins, fusion proteins, nucleic acid molecules, vectors, systems, complexes, and compositions of the invention.
In one embodiment, the delivery vehicle is a particle.
In one embodiment, the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microbubble, a gene gun, or a viral vector (e.g., replication defective retrovirus, lentivirus, adenovirus, or adeno-associated virus).
Host cells
The invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny comprising: cas proteins, fusion proteins, nucleic acid molecules, protein-nucleic acid complexes, activated CRISPR complexes, vectors, delivery compositions of the invention are described herein.
In certain embodiments, the cell is a prokaryotic cell.
In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, e.g., a non-human primate, bovine, ovine, porcine, canine, simian, rabbit, rodent (e.g., rat or mouse) cell. In certain embodiments, the cells are non-mammalian eukaryotic cells, such as cells of poultry birds (e.g., chickens), fish, or crustaceans (e.g., clams, shrimps). In certain embodiments, the cell is a plant cell, e.g., a cell of a monocot or dicot or a cell of a cultivated plant or a food crop such as tapioca, corn, sorghum, soybean, wheat, oat, or rice, e.g., an algae, tree, or production plant, fruit, or vegetable (e.g., a tree such as citrus, nut, eggplant, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).
In certain embodiments, the cell is a stem cell or stem cell line.
In certain instances, a host cell of the invention comprises a modification of a gene or genome that is not present in its wild type.
Gene editing method and application
The Cas protein, nucleic acid, the above composition, the above CIRSPR/Cas system, the above vector system, the above delivery composition or the above activated CRISPR complex or the above host cell of the present invention may be used for any one or any of several of the following uses: targeting and/or editing a target nucleic acid; cleaving double-stranded DNA, single-stranded DNA, or single-stranded RNA; nonspecific cleavage and/or degradation of collateral nucleic acids; nonspecifically cleaving the single-stranded nucleic acid; detecting nucleic acid; detecting nucleic acid in a target sample; editing the double-stranded nucleic acid specifically; base editing double-stranded nucleic acid; base editing single stranded nucleic acids. In other embodiments, it may also be used to prepare reagents or kits for any one or any of several of the uses described above.
The invention also provides the use of the Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition or activated CRISPR complex described above in gene editing, gene targeting or gene cleavage; alternatively, use in the preparation of a reagent or kit for gene editing, gene targeting or gene cleavage.
In one embodiment, the gene editing, gene targeting or gene cleaving is performed intracellularly and/or extracellularly.
The invention also provides a method of editing, targeting, or cleaving a target nucleic acid comprising contacting the target nucleic acid with the Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition, or activated CRISPR complex described above. In one embodiment, the method is editing, targeting, or cleaving a target nucleic acid inside or outside a cell.
The gene editing or editing target nucleic acids include modifying genes, knocking out genes, altering expression of gene products, repairing mutations, and/or inserting polynucleotides, gene mutations.
The editing may be performed in prokaryotic and/or eukaryotic cells.
In another aspect, the invention also provides the use of the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, or the activated CRISPR complex described above in nucleic acid detection, or in the preparation of a reagent or kit for nucleic acid detection.
In another aspect, the invention also provides a method of cleaving a single-stranded nucleic acid, the method comprising contacting a nucleic acid population with the Cas protein and the gRNA described above, wherein the nucleic acid population comprises a target nucleic acid and a plurality of non-target single-stranded nucleic acids, the Cas protein cleaving the plurality of non-target single-stranded nucleic acids.
The gRNA is capable of binding to the Cas protein.
The gRNA is capable of targeting the target nucleic acid.
The contacting may be inside a cell in vitro, ex vivo or in vivo.
Preferably, the cleavage of single-stranded nucleic acid is a nonspecific cleavage.
In another aspect, the invention also provides the use of the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition or the activated CRISPR complex described above for non-specific cleavage of single stranded nucleic acids, or for the preparation of a reagent or kit for non-specific cleavage of single stranded nucleic acids.
In another aspect, the invention also provides a kit for gene editing, gene targeting or gene cleavage comprising the Cas protein, gRNA, nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, the activated CRISPR complex or the host cell.
In another aspect, the invention also provides a kit for detecting a target nucleic acid in a sample, the kit comprising: (a) a Cas protein, or a nucleic acid encoding the Cas protein; (b) A guide RNA, or a nucleic acid encoding the guide RNA, or a precursor RNA comprising the guide RNA, or a nucleic acid encoding the precursor RNA; and (c) a single stranded nucleic acid detector that is single stranded and does not hybridize to the guide RNA.
It is known in the art that precursor RNAs can be cleaved or processed into the mature guide RNAs described above.
In another aspect, the invention provides the use of the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, the activated CRISPR complex, or the host cell described above in the preparation of a formulation or kit for:
(i) Gene or genome editing;
(ii) Target nucleic acid detection and/or diagnosis;
(iii) Editing a target sequence in a target locus to modify an organism or a non-human organism;
(iv) Treatment of disease;
(iv) Targeting the target gene.
Preferably, the gene or genome editing is performed in or out of a cell.
Preferably, the target nucleic acid detection and/or diagnosis is performed in vitro.
Preferably, the treatment of the disease is treatment of a condition caused by a defect in the target sequence in the target locus.
In another aspect, the invention provides a method of detecting a target nucleic acid in a sample, the method comprising contacting a sample with the Cas protein, a gRNA (guide RNA) comprising a region that binds to the Cas protein and a guide sequence that hybridizes to the target nucleic acid, and a single stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein cleaving single-stranded nucleic acid detector, thereby detecting a target nucleic acid; the single stranded nucleic acid detector does not hybridize to the gRNA.
Method for specifically modifying target nucleic acid
In another aspect, the invention also provides a method of specifically modifying a target nucleic acid, the method comprising: contacting a target nucleic acid with the Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition, or activated CRISPR complex.
The specific modification may occur in vivo or in vitro.
The specific modification may occur either intracellularly or extracellularly.
In some cases, the cell is selected from a prokaryotic cell or a eukaryotic cell, e.g., an animal cell, a plant cell, or a microbial cell.
In one embodiment, the modification refers to cleavage of the target sequence, e.g., single/double strand cleavage of DNA, or single strand cleavage of RNA.
In some cases, the method further comprises contacting the target nucleic acid with a donor polynucleotide, wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide is integrated into the target nucleic acid.
In one embodiment, the modification further comprises inserting an editing template (e.g., an exogenous nucleic acid) into the break.
In one embodiment, the method further comprises: contacting an editing template with the target nucleic acid, or delivering into a cell comprising the target nucleic acid. In this embodiment, the method repairs the disrupted target gene by homologous recombination with an exogenous template polynucleotide; in some embodiments, the repair results in a mutation, including an insertion, deletion, or substitution of one or more nucleotides of the target gene, in other embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.
Detection (non-specific cleavage)
In another aspect, the invention provides a method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, or the activated CRISPR complex and a single stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein cleaving single stranded nucleic acid detector, thereby detecting a target nucleic acid.
In the present invention, the target nucleic acid comprises ribonucleotides or deoxyribonucleotides; including single-stranded nucleic acids, double-stranded nucleic acids, e.g., single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA.
In one embodiment, the target nucleic acid is derived from a sample of a virus, bacterium, microorganism, soil, water source, human, animal, plant, or the like. Preferably, the target nucleic acid is a product of enrichment or amplification by methods such as PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM.
In one embodiment, the target nucleic acid is a viral nucleic acid, a bacterial nucleic acid, a specific nucleic acid associated with a disease, such as a specific mutation site or SNP site, or a nucleic acid that differs from a control; preferably, the virus is a plant virus or an animal virus, for example, papilloma virus, hepadnavirus, herpes virus, adenovirus, poxvirus, parvovirus, coronavirus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, mers-CoV.
In the present invention, the gRNA has a degree of match of at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% with the target sequence on the target nucleic acid.
In one embodiment, when the target sequence contains one or more characteristic sites (e.g., specific mutation sites or SNPs), the characteristic sites are perfectly matched to the gRNA.
In one embodiment, the detection method may comprise one or more grnas with different targeting sequences to different target sequences.
In the present invention, the single-stranded nucleic acid detector includes, but is not limited to, single-stranded DNA, single-stranded RNA, DNA-RNA hybrids, nucleic acid analogs, base modifications, single-stranded nucleic acid detectors containing abasic spacers, and the like; "nucleic acid analogs" include, but are not limited to: locked nucleic acids, bridged nucleic acids, morpholino nucleic acids, ethylene glycol nucleic acids, hexitol nucleic acids, threose nucleic acids, arabinose nucleic acids, 2' oxymethyl RNAs, 2' methoxyacetyl RNAs, 2' fluoro RNAs, 2' amino RNAs, 4' thio RNAs, and combinations thereof, including optional ribonucleotide or deoxyribonucleotide residues.
In the present invention, the detectable signal is realized by: visual-based detection, sensor-based detection, color detection, fluorescent signal-based detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase change/dispersion, electrochemical detection, and semiconductor-based detection.
In the present invention, it is preferable that both ends of the single-stranded nucleic acid detector are provided with a fluorescent group and a quenching group, respectively, and that the single-stranded nucleic acid detector may exhibit a detectable fluorescent signal when cleaved. The fluorescent group is selected from one or more of FAM, FITC, VIC, JOE, TET, CY, CY5, ROX, texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.
In other embodiments, the 5 'end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different labeling molecules, and colloidal gold test results before the single-stranded nucleic acid detector is cleaved by the Cas protein and after the single-stranded nucleic acid detector is cleaved by the Cas protein are detected by a colloidal gold detection mode; the single-stranded nucleic acid detector will exhibit different color development results on the detection line and the quality control line of colloidal gold before being cleaved by Cas protein and after being cleaved by Cas protein.
In some embodiments, the method of detecting a target nucleic acid may further comprise comparing the level of the detectable signal to a reference signal level, and determining the amount of the target nucleic acid in the sample based on the level of the detectable signal.
In some embodiments, the method of detecting a target nucleic acid may further comprise using RNA reporter nucleic acid and DNA reporter nucleic acid (e.g., fluorescent colors) on different channels, and sampling based on combining (e.g., using a minimum or product) the levels of the detectable signals by measuring the signal levels of the RNA and DNA reporter molecules, and determining the levels of the detectable signals by measuring the amounts of target nucleic acid in the RNA and DNA reporter molecules.
In one embodiment, the target gene is present in a cell.
In one embodiment, the cell is a prokaryotic cell.
In one embodiment, the cell is a eukaryotic cell.
In one embodiment, the cell is an animal cell.
In one embodiment, the cell is a human cell.
In one embodiment, the cell is a plant cell, such as a cell of a cultivated plant (e.g., cassava, maize, sorghum, wheat, or rice), algae, tree, or vegetable.
In one embodiment, the target gene is present in an in vitro nucleic acid molecule (e.g., a plasmid).
In one embodiment, the target gene is present in a plasmid.
Definition of terms
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Further, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA, etc., as used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
Cas proteins
In the present invention, cas protein, cas enzyme, cas effector protein may be used interchangeably; the present inventors have first discovered and identified a Cas effector protein having an amino acid sequence selected from the group consisting of:
(i) SEQ ID No.1 or SEQ ID No.4 or SEQ ID No.7;
(ii) A sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) as compared to the sequence set forth in SEQ ID No.1 or SEQ ID No.4 or SEQ ID No.7; or (b)
(iii) A sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID No.1 or SEQ ID No.4 or SEQ ID No. 7.
Nucleic acid cleavage or cleavage nucleic acids herein include DNA or RNA cleavage (Cis cleavage), cleavage of DNA or RNA in a side branch nucleic acid substrate (single-stranded nucleic acid substrate) in a target nucleic acid produced by a Cas enzyme described herein (i.e., non-specific or non-targeted, trans cleavage). In some embodiments, the cleavage is a double-stranded DNA break. In some embodiments, the cleavage is a single-stranded DNA cleavage or a single-stranded RNA cleavage.
CRISPR system
As used herein, the term "regularly clustered, spaced short palindromic repeats (CRISPR) -CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" is used interchangeably and has the meaning commonly understood by those skilled in the art, which generally comprises transcripts or other elements related to the expression of a CRISPR-associated ("Cas") gene, or transcripts or other elements capable of directing the activity of the Cas gene.
CRISPR/Cas complexes
As used herein, the term "CRISPR/Cas complex" refers to a complex formed by directing RNA (guide RNA) or mature crRNA to bind to a Cas protein, comprising a direct repeat sequence that hybridizes to a target sequence and binds to a Cas protein, which complex is capable of recognizing and cleaving a polynucleotide that hybridizes to the guide RNA or mature crRNA.
Guide RNA (guide RNA, gRNA)
As used herein, the terms "guide RNA", "mature crRNA", "guide sequence" are used interchangeably and have the meaning commonly understood by those skilled in the art. In general, the guide RNA can comprise, consist essentially of, or consist of a direct repeat (direct repeat) and a guide sequence.
In certain instances, the guide sequence is any polynucleotide sequence that has sufficient complementarity to a target sequence to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence. In one embodiment, the degree of complementarity between a guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% when optimally aligned. It is within the ability of one of ordinary skill in the art to determine the optimal alignment. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, the Smith-Waterman algorithm (Smith-Waterman), bowtie, geneious, biopython, and SeqMan in ClustalW, matlab.
Target sequence
"target sequence" refers to a polynucleotide targeted by a guide sequence in a gRNA, e.g., a sequence that has complementarity to the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote the formation of a CRISPR/Cas complex (including Cas proteins and grnas). Complete complementarity is not necessary so long as sufficient complementarity exists to cause hybridization and promote the formation of a CRISPR/Cas complex.
The target sequence may comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located either inside or outside the cell. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located within an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. Sequences or templates that can be used for recombination into a target locus comprising the target sequence are referred to as "editing templates" or "editing polynucleotides" or "editing sequences". In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.
In the present invention, a "target sequence" or "target polynucleotide" or "target nucleic acid" may be any polynucleotide that is endogenous or exogenous to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide may be a polynucleotide that is present in the nucleus of a eukaryotic cell. The target polynucleotide may be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or unwanted DNA). In some cases, the target sequence should be related to the Protospacer Adjacent Motif (PAM).
Single-stranded nucleic acid detector
The single-stranded nucleic acid detector according to the present invention means a detector comprising a sequence of 2 to 200 nucleotides, preferably 2 to 150 nucleotides, preferably 3 to 100 nucleotides, preferably 3 to 30 nucleotides, preferably 4 to 20 nucleotides, more preferably 5 to 15 nucleotides. Preferably a single-stranded DNA molecule, a single-stranded RNA molecule or a single-stranded DNA-RNA hybrid.
The single-stranded nucleic acid detector comprises different reporter groups or marker molecules at both ends, which do not exhibit a reporter signal when in an initial state (i.e., not cleaved), and which exhibit a detectable signal when cleaved, i.e., a detectable distinction between cleaved and pre-cleaved.
In one embodiment, the reporter or marker molecule comprises a fluorophore and a quencher, wherein the fluorophore is selected from one or more of FAM, FITC, VIC, JOE, TET, CY, CY5, ROX, texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.
In one embodiment, the single stranded nucleic acid detector has a first molecule (e.g., FAM or FITC) attached to the 5 'end and a second molecule (e.g., biotin) attached to the 3' end. The reaction system containing the single-stranded nucleic acid detector is matched with a flow strip to detect target nucleic acid (preferably, a colloidal gold detection mode). The flow strip is designed with two capture lines, with an antibody binding to a first molecule (i.e., a first molecular antibody) at the sample contact end (colloidal gold), an antibody binding to the first molecular antibody at the first line (control line), and an antibody binding to a second molecule (i.e., a second molecular antibody, such as avidin) at the second line (test line). When the reaction flows along the strip, the first molecular antibody binds to the first molecule carrying the cleaved or uncleaved oligonucleotide to the capture line, the cleaved reporter will bind to the antibody of the first molecular antibody at the first capture line, and the uncleaved reporter will bind to the second molecular antibody at the second capture line. Binding of the reporter group at each line will result in a strong readout/signal (e.g., color). As more reporter is cut, more signal will accumulate at the first capture line and less signal will appear at the second line. In certain aspects, the invention relates to the use of a flow strip as described herein for detecting nucleic acids. In certain aspects, the invention relates to a method of detecting nucleic acids using a flow strip as defined herein, e.g. a (lateral) flow test or a (lateral) flow immunochromatographic assay. In certain aspects, the molecules in the single stranded nucleic acid detector may be interchanged or the positions of the molecules may be changed, so long as the reporting principle is the same or similar to that of the present invention, and the modified manner is also included in the present invention.
The detection method provided by the invention can be used for quantitative detection of target nucleic acid to be detected. The quantitative detection index can be quantified according to the signal intensity of the reporter group, such as the luminous intensity of the fluorescent group, the width of the color-developing strip, and the like.
Wild type
As used herein, the term "wild-type" has the meaning commonly understood by those skilled in the art, which refers to a typical form of an organism, strain, gene, or a characteristic that, when it exists in nature, differs from a mutant or variant form, which may be isolated from a source in nature and not intentionally modified by man.
Derivatization
As used herein, the term "derivatization" refers to a chemical modification of an amino acid, polypeptide, or protein in which one or more substituents have been covalently attached to the amino acid, polypeptide, or protein. Substituents may also be referred to as side chains.
A derivatized protein is a derivative of the protein, in general, derivatization of the protein does not adversely affect the desired activity of the protein (e.g., binding to a guide RNA, endonuclease activity, binding to a specific site of a target sequence under the guidance of a guide RNA and cleavage activity), that is, the derivative of the protein has the same activity as the protein.
Derivatizing proteins
Also referred to as "protein derivatives" refers to modified forms of a protein, for example, wherein one or more amino acids of the protein may be deleted, inserted, modified and/or substituted.
Non-naturally occurring
As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and refer to human involvement. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component to which it is associated in nature or as found in nature.
Orthologs (orthologs)
As used herein, the term "ortholog" has a meaning commonly understood by those skilled in the art. As a further guidance, an "ortholog" of a protein as described herein refers to a protein belonging to a different species that performs the same or similar function as the protein as its ortholog.
Identity of
As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum 62 matrix or PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.
Carrier body
The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule linked thereto. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; a nucleic acid molecule comprising one or more free ends, free ends (e.g., circular); a nucleic acid molecule comprising DNA, RNA, or both; and other diverse polynucleotides known in the art. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. A vector may be introduced into a host cell to thereby produce a transcript, protein, or peptide, including from a protein, fusion protein, isolated nucleic acid molecule, or the like (e.g., a CRISPR transcript, such as a nucleic acid transcript, protein, or enzyme) as described herein. A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.
One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA fragments may be inserted, for example, by standard molecular cloning techniques.
Another type of vector is a viral vector in which a virus-derived DNA or RNA sequence is present in a vector used to package a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). Viral vectors also comprise polynucleotides carried by a virus for transfection into a host cell. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced.
Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors".
Host cells
As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as e.g. escherichia coli or bacillus subtilis, eukaryotic cells such as microbial cells, fungal cells, animal cells and plant cells.
Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of expression, and the like.
Regulatory element
As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly U sequences), the detailed description of which may be found in goldel (Goeddel), gene expression techniques: methods of enzymology (GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY) 185, academic Press (Academic Press), san Diego (San Diego), calif. (1990). In some cases, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those sequences that direct expression of the nucleotide sequence in only certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). In some cases, regulatory elements may also direct expression in a time-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In certain instances, the term "regulatory element" encompasses enhancer elements, such as WPRE; a CMV enhancer; the R-U5' fragment in the LTR of HTLV-I (mol. Cell. Biol., volume 8 (1), pages 466-472, 1988), the SV40 enhancer, and the intron sequence between exons 2 and 3 of rabbit beta-globin (Proc. Natl. Acad. Sci. USA., volume 78 (3), pages 1527-31, 1981).
Promoters
As used herein, the term "promoter" has a meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that is capable of initiating expression of a downstream gene. Constitutive (constitutive) promoters are nucleotide sequences of: when operably linked to a polynucleotide encoding or defining a gene product, it results in the production of the gene product in the cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in a cell, essentially only when an inducer corresponding to the promoter is present in the cell. Tissue specific promoters are nucleotide sequences that: when operably linked to a polynucleotide encoding or defining a gene product, it results in the production of the gene product in the cell substantially only if the cell is a cell of the tissue type to which the promoter corresponds.
NLS
A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for introduction into the nucleus by nuclear transport, i.e., a protein with NLS is transported to the nucleus. Typically, NLS contains positively charged Lys or Arg residues exposed at the protein surface. Exemplary nuclear localization sequences include, but are not limited to, NLS from: SV40 large T antigen, EGL-13, c-Myc, and TUS proteins. In some embodiments, the NLS comprises PKKKRKV sequence. In some embodiments, the NLS comprises the AVKRPAATKKAGQAKKKKLD sequence. In some embodiments, the NLS comprises the PAAKRVKLD sequence. In some embodiments, the NLS comprises the MSRRRKANPTKLSENAKKLAKEVEN sequence. In some embodiments, the NLS comprises the KLKIKRPVK sequence. Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP A1, the sequences KIPIK and PY-NLS in yeast transcription repressor Mat. Alpha.2.
Operatively connected to
As used herein, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
Complementarity and method of detecting complementary
As used herein, the term "complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of a conventional watson-crick or other non-conventional type. Percent complementarity means the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "fully complementary" means that all consecutive residues of one nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in one second nucleic acid sequence. "substantially complementary" as used herein refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.
Stringent conditions
As used herein, "stringent conditions" for hybridization refers to conditions under which a nucleic acid having complementarity to a target sequence hybridizes predominantly to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are typically sequence-dependent and will vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.
Hybridization
The term "hybridization" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA, DNA) comprises a nucleotide sequence that enables it to bind non-covalently, i.e., form base pairs and/or G/U base pairs with another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to the complementary nucleic acid), "anneal" or "hybridize".
Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between bases. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. Typically, the hybridizable nucleic acid is 8 nucleotides or more in length (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).
It will be appreciated that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to specifically hybridize. Polynucleotides may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region in a target nucleic acid sequence to which it hybridizes.
Hybridization of the target sequence to the gRNA represents that at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the target sequence and the nucleic acid sequence of the gRNA can hybridize to form a complex; or at least 12, 15, 16, 17, 18, 19, 20, 21, 22 or more bases of the nucleic acid sequence representing the target sequence and the gRNA may be complementarily paired and hybridized to form a complex.
Expression of
As used herein, the term "expression" refers to a process whereby a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and/or a process whereby the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.
Joint
As used herein, the term "linker" refers to a linear polypeptide formed from multiple amino acid residues joined by peptide bonds. The linker of the invention may be an amino acid sequence that is synthesized artificially, or a naturally occurring polypeptide sequence, such as a polypeptide having the function of a hinge region. Such linker polypeptides are well known in the art (see, e.g., holliger, P. Et al (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; poljak, R.J. Et al (1994) Structure 2:1121-1123).
Treatment of
As used herein, the term "treating" refers to treating or curing a disorder, delaying the onset of symptoms of a disorder, and/or delaying the progression of a disorder.
A subject
As used herein, the term "subject" includes, but is not limited to, various animals, plants, and microorganisms.
Animals
Such as mammals, e.g., bovine, equine, ovine, porcine, canine, feline, lagomorph (e.g., mice or rats), non-human primate (e.g., macaque or cynomolgus) or human. In certain embodiments, the subject (e.g., human) has a disorder (e.g., a disorder resulting from a disease-related gene defect).
Plants and methods of making the same
The term "plant" is understood to mean any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichoke, broccoli, sesame seed, leek, asparagus, lettuce (e.g., head lettuce, leaf lettuce), cabbage (bok choy), yellow arrowroot, melons (e.g., melon, watermelon, columbian melon (crenhaw), white melon, cantaloupe), rape crops (e.g., cabbage, broccoli, chinese cabbage, kohlrabi, chinese cabbage), artichoke, carrot, cabbage (napa), okra, onion, celery, parsley, chick pea, parsnip, chicory, pepper, potato, cucurbit (e.g., zucchini, cucumber, zucchini, melon, pumpkin), radish, dried onion, turnip cabbage, purple eggplant (also known as eggplant), salon, chicory, shallot, chicory, garlic, spinach, green onion, melon, green leafy vegetables (greens), beet (sugar beet and fodder beet), sweet potato, lettuce, horseradish, tomato, turnip, spice; fruit and/or vining crops, such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, oranges, blueberries, boysenberries (boysenberries), redberries, currants, rowfruits, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwi fruits, persimmons, pomegranates, pineapple, tropical fruits, pome fruits, melons, mangoes, papaya, and litchis; field crops, such as clover, alfalfa, evening primrose, white mango, corn/maize (forage maize, sweet maize, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereal crops (barley, oat, rye, wheat, etc.), sorghum, tobacco, kapok, leguminous plants (beans, lentils, peas, soybeans), oleaginous plants (rape, mustard, poppy, olives, sunflower, coconut, castor oil plants, cocoa beans, groundnut), arabidopsis, fibrous plants (cotton, flax, hemp, jute), camphorridae (cinnamon, camphordons), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and/or flower bed plants, such as flowering plants, cactus, fleshy plants and/or ornamental plants, and trees, such as forests (broadleaf and evergreen trees, e.g., conifers), fruit trees, ornamental trees, and nut-bearing trees, and shrubs and other seedlings.
Advantageous effects of the application
The application discovers a novel Cas enzyme, and Blast results show that the Cas enzyme has lower consistency with the reported Cas enzyme, can show nuclease activity in vivo and in vitro, and has wide application prospect.
Embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present application and are not to be construed as limiting the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.
Drawings
FIG. 1 shows a graph of fluorescence results of Cas-sf0009 for nucleic acid detection.
FIG. 2 shows fluorescence results of Cas-sf1782 and Cas-sf2431 for nucleic acid detection.
FIG. 3 PAM structure of Cas-sf 0009.
FIG. 4 cleavage results of double-stranded nucleic acid by Cas-sf 0009.
FIG. 5 results of Cas-sf0009 versus NTS/TS cleavage efficiency.
FIG. 6. Position of Cas-sf0009 cleavage of target nucleic acid.
The PAM structure of cas-sf 1782.
FIG. 8 cleavage results of double-stranded nucleic acid by Cas-sf 1782.
The PAM structure of cas-sf 2431.
Sequence information
SEQ ID No. | Description of the invention |
1 | Amino acid sequence of Cas-sf0009 |
2 | Nucleic acid sequence encoding Cas-sf0009 |
3 | Humanized Cas-sf0009 nucleic acid sequences |
4 | Amino acid sequence of Cas-sf1782 |
5 | Nucleic acid sequence encoding Cas-sf1782 |
6 | Humanized Cas-sf1782 nucleic acid sequences |
7 | Amino acid sequence of Cas-sf2431 |
8 | Nucleic acid sequence encoding Cas-sf2431 |
9 | Humanized Cas-sf2431 nucleic acid sequences |
10 | Cas-sf0009-DR |
11 | Cas-sf1782-DR |
12 | Cas-sf2431-DR |
Detailed Description
The following examples are only intended to illustrate the invention and are not intended to limit it. The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, for the conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention, reference may be made to Sambrook (Sambrook), friech (Fritsch) and manitis (Maniatis), molecular cloning: laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), edit 2 (1989); the handbook of contemporary molecular biology (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (edited by f.m. ausubel (f.m. ausubel) et al, (1987)); series (academic publishing company) of methods in enzymology (METHODS IN ENZYMOLOGY): PCR 2: practical methods (PCR 2:A PRACTICAL APPROACH) (m.j. Maxfresen (m.j. Macpherson), b.d. black ms (b.d. hames) and g.r. taylor (1995)), harlow and Lane (Lane) edits (1988), antibodies: laboratory Manual (ANTIBODIES, ALABORATORY Manual), animal cell CULTURE (ANIMAL CELL CULTURE) (R.I. French Lei Xieni (R.I. Freshney) eds. (1987)).
In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.
EXAMPLE 1 Cas protein acquisition
The inventors analyzed the macrogenome of the uncultured, and identified four new Cas enzymes by redundancy removal, protein cluster analysis. Blast results show that the Cas protein has lower sequence identity with the reported Cas protein, and the Cas protein is named as Cas-sf0009, cas-sf1782 and Cas-sf2431 in the invention; analysis of the evolutionary tree shows that the Cas protein belongs to Cas12j family proteins; the amino acid sequence, the coding nucleic acid sequence and the nucleic acid sequence of the protein after human codon optimization are shown as follows:
SEQ ID No. | description of the invention |
1 | Amino acid sequence of Cas-sf0009 |
2 | Nucleic acid sequence encoding Cas-sf0009 |
3 | Humanized Cas-sf0009 nucleic acid sequences |
4 | Amino acid sequence of Cas-sf1782 |
5 | Nucleic acid sequence encoding Cas-sf1782 |
6 | Humanized Cas-sf1782 nucleic acid sequences |
7 | Amino acid sequence of Cas-sf2431 |
8 | Nucleic acid sequence encoding Cas-sf2431 |
9 | Humanized Cas-sf2431 nucleic acid sequences |
Analysis of the homologous repeat sequence of the gRNA corresponding to the protein shows that:
the homologous repeated sequence of the gRNA corresponding to the Cas-sf0009 protein is
GUGCAGCCUAUUGAUUAUUCUCGAUAGGCAUAGCAC(SEQ ID No.10);
The homologous repeated sequence of the gRNA corresponding to the Cas-sf1782 protein is
CUAACUGUGUGAGCUUCUAACCGAAGCUAAUGACAC(SEQ ID No.11);
The homologous repeated sequence of the gRNA corresponding to the Cas-sf2431 protein is
GUGUCAGAAGACUUAUAAAAUGAGUCUUAUUGUGAC(SEQ ID No.12)。
Example 2 application of Cas-sf0009 protein in nucleic acid detection
This example demonstrates the trans-cleavage activity of Cas-sf0009 by in vitro assays. The Cas-sf0009 protein is guided in this example to recognize and bind to the target nucleic acid using a gRNA that can be paired with the target nucleic acid; subsequently, the Cas-sf0009 protein triggers trans cleavage activity on any single stranded nucleic acid, thereby cleaving the single stranded nucleic acid detector in the system; fluorescent groups and quenching groups are respectively arranged at two ends of the single-stranded nucleic acid detector, and if the single-stranded nucleic acid detector is cut, fluorescence is excited; in other embodiments, both ends of the single-stranded nucleic acid detector may be provided with a label that can be detected by colloidal gold.
In this example, the target nucleic acid is selected to be single-stranded DNA, ssDNA: TCAAGCTGGTTCAATCTGTCAAGCAGCAGCAAAGCAAGAGCAGC;
gRNA sequence GUGCAGCCUAUUGAUUAUUCUCGAUAGGCAUAGCACUUGCUGCUGCUUGACAGAUU(the underlined region is the targeting region);
the sequence of the single-stranded nucleic acid detector is FAM-TTATT-BHQ1;
the following reaction system is adopted: cas-sf0009 final concentration was 50nM, gRNA final concentration was 50nM, target nucleic acid final concentration was 50nM, single-stranded nucleic acid detector final concentration was 200nM. FAM fluorescence/30 s was read by incubation at 37 ℃. Control 1 did not add target nucleic acid and control 2 did not add gRNA.
As shown in FIG. 1, cas-sf0009 was able to detect single stranded nucleic acids in the cleavage system and rapidly report fluorescence in the presence of target nucleic acids and gRNA compared to the control. The above experiments reflect that Cas-sf0009 can be used for detection of target nucleic acids in combination with single stranded nucleic acid detectors. In FIG. 1, 1 is the experimental result of adding the target nucleic acid to Cas-sf0009, 2 is the control group 1 to which the target nucleic acid is not added to Cas-sf0009, and 3 is the control group 2 to which the gRNA is not added to Cas-sf 0009.
Example 3 application of Cas-sf1782 protein in nucleic acid detection
Referring to the method of example 2, this example demonstrates the trans-cleavage activity of Cas-sf1782 by in vitro assays.
In this example, the target nucleic acid is selected to be single-stranded DNA, ssDNA: TCAAGCTGGTTCAATCTGTCAAGCAGCAGCAAAGCAAGAGCAGC;
gRNA sequence CUAACUGUGUGAGCUUCUAACCGAAGCUAAUGACACUUGCUGCUGCUUGACAGAUU(the underlined region is the targeting region);
the sequence of the single-stranded nucleic acid detector is FAM-TTATT-BHQ1;
the following reaction system is adopted: cas-sf1782 final concentration was 50nM, gRNA final concentration was 50nM, target nucleic acid final concentration was 50nM, single-stranded nucleic acid detector final concentration was 200nM. FAM fluorescence/20 s was read by incubation at 37 ℃. The control group had no target nucleic acid added.
As shown in FIG. 2, cas-sf1782 can detect single stranded nucleic acid in the cleavage system in the presence of target nucleic acid, reporting fluorescence rapidly, as compared to a control without target nucleic acid. The above experiments reflect that Cas-sf1782 can be used for detection of target nucleic acids in combination with single stranded nucleic acid detectors. In FIG. 2, 1 is the experimental result of adding the target nucleic acid to Cas-sf1782, and 3 is the control group to which the target nucleic acid is not added to Cas-sf 1782.
Example 4 application of Cas-sf2431 protein in nucleic acid detection
Referring to the method of example 2, this example demonstrates the trans-cleavage activity of Cas-sf2431 by in vitro assays.
In this example, the target nucleic acid is selected to be single-stranded DNA, ssDNA: TCAAGCTGGTTCAATCTGTCAAGCAGCAGCAAAGCAAGAGCAGC;
gRNA sequence GUGUCAGAAGACUUAUAAAAUGAGUCUUAUUGUGAC UUGCUGCUGCUUGACAGAUU(the underlined region is the targeting region);
the sequence of the single-stranded nucleic acid detector is FAM-TTATT-BHQ1;
the following reaction system is adopted: cas-sf2431 final concentration was 50nM, gRNA final concentration was 50nM, target nucleic acid final concentration was 50nM, single-stranded nucleic acid detector final concentration was 200nM. FAM fluorescence/20 s was read by incubation at 37 ℃. The control group had no target nucleic acid added.
As shown in FIG. 2, cas-sf2431 is capable of detecting single stranded nucleic acid in the cleavage system in the presence of target nucleic acid, reporting fluorescence, as compared to a control without target nucleic acid. The above experiments show that Cas-sf2431 can be used for detection of target nucleic acids in combination with single stranded nucleic acid detectors, but Cas-sf2431 has significantly weaker detection effect than other Cas proteins described above. In FIG. 2, 2 is the experimental result of adding the target nucleic acid to the Cas-sf2431, and 4 is the control group of the Cas-sf2431 without adding the target nucleic acid.
EXAMPLE 5 PAM identification of Cas-sf0009 protein
Construction of Cas-sf0009 protein expression plasmid: the nucleic acid sequence is subjected to genetic synthesis after being subjected to humanized codon optimization, and is connected with an escherichia coli expression vector PeT28 (a) +vector. The addition of the JM23119 promoter to vector PeT28 (a) + -Cas-sf0009 initiates transcription of Cas-sf0009 CrRNA. Forming a carrier: peT28 (a) + -Cas-sf0009-JM23119-crRNA, crRNA sequence: GUGCAGCCUAUUGAUUAUUCUCGAUAGGCAUAGCAC UCCCCUACGUGCUGCUGAAGUUGCUnderlined is the target sequence; construction of PAM library: synthetic sequence CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTCAGCGTTCNNNNNNTCCCCTACGTGCTGCTGAAGTTGCCCGCAA, N is a random deoxynucleotide, underlined is the target sequence. The vector was filled in with Klenow enzyme and ligated into the pacyc184 vector. And (3) converting escherichia coli, and extracting plasmids to form a PAM library.
PAM library subtraction experiment: preparation of competence: BL21 (DE 3) -PeT28 (a) + -Cas-sf0009-JM23119-crRNA. PAM library plasmid transformation competence: BL21 (DE 3) -PeT28 (a) + -Cas-sf0009-JM23119-crRNA is coated on LB plates containing kanamycin and chloramphenicol, bacterial cells are collected after overnight culture at 37 ℃, the bacterial concentration is adjusted to OD 600.6-0.8, IPTG 0.2mM is added, and induction is performed for 4 hours at 37 ℃. FastPure EndoFree Plasmid Maxi Kit (vazyme) plasmid extraction was performed to obtain post-subtraction PAM libraries. Primer: PAM-F: GGTCTTCGGTTTCCGTGTT; PAM-R: TGGCGTTGACTCTCAGTCAT. The control samples were obtained by PCR using 30 ng/. Mu.L of plasmid (PAM library) as a template primer, and the experimental samples were obtained by PCR using 30 ng/. Mu.L of plasmid (PAM library after subtraction) as a template. The control and experimental samples were submitted to second generation sequencing for data analysis using weblog mapping and Cas-sf0009 (upon in vivo cleavage) identified a PAM structure of 5'-NTT-3', where n=a/T/C/G.
EXAMPLE 6 application of Cas-sf0009 protein in double stranded nucleic acid editing
Examples the cis-cleavage activity of double stranded DNA of Cas-sf0009 was tested in vitro. In this example, the Cas-sf0009 protein is recognized and bound to the target nucleic acid by using the gRNA that can pair with the target nucleic acid, thereby cleaving the target nucleic acid in the system, and performing agarose electrophoresis detection on the cleaved target nucleic acid.
In this example, the target nucleic acid was selected to be double-stranded DNA (plasmid), 5space 1-PAM, which has the sequence:ligating into Vector T-Vector-pEASY-Blunt Simple Cloning Vector; the italic part is PAM sequence, n=a/T/C/G, the underlined region is the targeting region.
gRNA:Cas-sf0009-5spacer1:
GUGCAGCCUAUUGAUUAUUCUCGAUAGGCAUAGCACUCCCCUACGUGCUGCUGAAG(underlined region is the targeting region)
The following reaction system is adopted: 20 μl system, buffer: tris-HAC 40mM, mg (AC) 2 30mM, BSA 120. Mu.g/ml, DTT 12mM, pH7.0, final Cas-sf0009 concentration of 100nM, final gRNA concentration of 200nM, final double-stranded target nucleic acid concentration of 5 ng/. Mu.L. Incubation at 37℃for 1h and 85℃for 20min. The cleavage products were subjected to agarose electrophoresis to detect Cas-sf0009 cleavage capacity. The experimental group added Cas-sf0009 protein, gRNA and target nucleic acid, and the control group (CK) did not add gRNA.
The results are shown in fig. 4, where Cas-sf0009 is able to cleave double stranded nucleic acids in the system in the experimental group compared to the control without gRNA, and PAM showing a distinct cleavage band includes 5'-ATD-3',5'-TTN-3',5'-STT-3', where n=a/T/C/G, s=c/G, d=a/G/T. This suggests that Cas-sf0009 (when cleaved in vitro) can be used for cleavage and editing of PAM as a double stranded target nucleic acid including, but not limited to, 5'-ATD-3',5'-TTN-3',5 '-STT-3'.
EXAMPLE 7 cleavage Property of Cas-sf0009 protein-NTS/TS cleavage efficiency
This example detects in vitro the cleavage efficiency of Cas-sf0009 on target nucleotide double-stranded DNA complementary strand (TS), non-complementary strand (NTS). 5'6-FAM labeled non-complementary strand (NTS), 5' ROX labeled complementary strand (TS), gRNA directs the recognition and binding of Cas-sf0009 protein to the target nucleic acid, thereby cleaving the target nucleic acid in the system, and capillary electrophoresis detection of the cleaved target nucleic acid (ABI 3730xl genetic Analyzer). The DNA fragments migrate from the cathode to the anode in the gel, are arranged according to the length of the fragments, when migrating to the scanning window of the laser scanner at the anode end, the fluorescent dye is excited to emit light with a certain wavelength, the light is recorded according to the fluorescence intensity, the electrophoresis track of each DNA fragment with the fluorescent dye is recorded according to the actual time of each DNA fragment passing through the laser scanning window, and each fragment is represented by a fluorescence absorption peak. The higher the peak, the more the fragment amount; the time at which the peak occurs has a direct relationship with the fragment size, the smaller the fragment the earlier the peak occurs. The size of the uncleaved NTS fragment of Cas-sf0009 in the FAM fluorescence channel was 380nt, and the post-NTS fragment of Cas-sf0009 was about 126nt. The size of the uncleaved TS fragment of Cas-sf0009 in the ROX fluorescence channel is 380nt, and the size of the fragment after cleavage of TS by Cas-sf0009 is about 254nt. Fragment cutting efficiency calculation formula: cleavage efficiency = cleavage peak area/(cleavage peak area + uncleaved peak area).
In this example, the target nucleic acid was selected to be double-stranded DNA (PCR product), and the primer:
XQ0001-5FAM:GTATGTTGTGTGGAATTGTG 5'6-FAM;
XQ0002-5ROX:GCTGCGCGTAACCACCACAC 5'ROX
the sequence of the amplified product is as follows:
the italic portion is PAM sequence and the underlined region is the targeting region.
gRNA:Cas-sf0009-5spacer1:
GUGCAGCCUAUUGAUUAUUCUCGAUAGGCAUAGCACUCCCCUACGUGCUGCUGAAG(underlined region is the targeting region)
The following reaction system is adopted: 20 μl system, buffer: tris-HAC 40mM, mg (AC) 2 30mM, BSA 120. Mu.g/ml, DTT 12mM, pH7.0, cas-sf0009 50nM, gRNA100nM, double-stranded target nucleic acid 1. Mu.L (PCR product). Incubation at 37deg.C for 5min, 15min, 30min, 60min, and 85 deg.C for 20min. Capillary electrophoresis detects (ABI 3730xl genetic analyzer) the products in FAM, ROX channels. The software Gene mapper 4.1 performs data analysis to calculate the cleavage efficiencies of NTS and TS.
The results are shown in FIG. 5: cas-sf0009 preferentially cleaves NTS when cleaving a target nucleic acid.
EXAMPLE 8 cleavage Properties of Cas-sf0009 protein cleavage position
This example measures the cleavage site of Cas-sf0009 protein on the complementary strand (TS), non-complementary strand (NTS) of double-stranded target nucleic acid by in vitro detection. The Cas-sf0009 protein is guided in this example by gRNA to recognize and bind to double stranded target nucleic acids; cas proteins trigger cis cleavage activity on double-stranded target nucleic acids, thereby cleaving double-stranded target nucleic acids in the system. And (3) adding the adaptor with the T after the supplement A to the double-stranded target nucleic acid after cutting, and carrying out sanger sequencing after PCR enrichment on the connected product.
In this example, the target nucleic acid was selected to be double-stranded DNA (plasmid), the sequence:the Vector T-Vector-pEASY-Blunt Simple Cloning Vector was ligated, the italic was PAM sequence and the underlined was the targeting region.
gRNA:Cas-sf0009-5spacer1:
GUGCAGCCUAUUGAUUAUUCUCGAUAGGCAUAGCACUCCCCUACGUGCUGCUGAAG(underlined regions are targeting regions).
The following reaction system is adopted: 50 μl system, buffer: tris-HAC 40mM, mg (AC) 2 30mM, BSA 120. Mu.g/ml, DTT 12mM, pH7.0, cas-sf0009 100nM, gRNA 250nM, double-stranded target nucleic acid 10 ng/. Mu.L (plasmid). Cas protein, gRNA, incubation for 10min at 25 ℃; adding double-stranded target nucleic acid, incubating for 1h at 37 ℃ and incubating for 5min at 85 ℃; 50uL 2X Taq DNA Polymerase Mix (Nuozan) (1:1) is added into the system, and the reaction is carried out for 30min at 72 ℃; the reaction liquid is subjected to liquid recovery; the recovery liquid was added to 2. Mu.L of annealed primer 2. Mu.M (TK-117: CGGCATTCCTGAACCGTCTCTTTCCGATCT, TK-111: GATCGGAAGAGCGGTTCAGCAGGAATGCCG), T4 (NEB) ligase at 22℃for 1h. 10. Mu.L of ligation product was taken, primer S1-PAM-after: ACTCAGCGGCATTCCTGCTGAACCGC, PQ0275-F: CCGTATTACCGCCTTTGAG,2X Taq DNA Polymerase Mix (Northenzan) were subjected to PCR reactions. The PCR products were subjected to sanger sequencing.
As a result, as shown in FIG. 6, the Cas-sf0009 protein cleaves the target nucleic acid at a position between 20-21nt of NTS and 23-24 of TS. That is, the cleavage site of the complementary strand of Cas-sf0009 to the target sequence is between 23nt and 24nt at the 5 'end of the PAM complementary sequence, the cleavage site of the non-complementary strand of Cas-sf0009 to the target sequence is between 20nt and 21nt at the 3' end of the PAM sequence, and the gRNA directs Cas-sf0009 protein to recognize and bind to the complementary strand, which is the DNA strand paired with the complementary strand.
EXAMPLE 9 PAM identification of Cas-sf1782 protein
Construction of Cas-sf1782 protein expression plasmid: the nucleic acid sequence is subjected to genetic synthesis after being subjected to humanized codon optimization, and is connected with an escherichia coli expression vector PeT28 (a) +vector. The addition of the JM23119 promoter to vector PeT28 (a) + -Cas-sf1782 initiates transcription of Cas-sf1782 CrRNA. Forming a carrier: peT28 (a) + -Cas-sf1782-JM23119-crRNA, crRNA sequence: CUAACUGUGUGAGCUUCUAACCGAAGCUAAUGACACUCCCCUACGUGCUGCUGAAGUUGCUnderlined is the target sequence; construction of PAM library: synthetic sequence CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTCAGCGTTCNNNNNNTCCCCTACGTGCTGCTGAAGTTGCCCGCAA, N is a random deoxynucleotide, underlined is the target sequence. The vector was filled in with Klenow enzyme and ligated into the pacyc184 vector. And (3) converting escherichia coli, and extracting plasmids to form a PAM library.
PAM library subtraction experiment: preparation of competence: BL21 (DE 3) -PeT28 (a) + -Cas-sf1782-JM23119-crRNA. PAM library plasmid transformation competence: BL21 (DE 3) -PeT28 (a) + -Cas-sf1782-JM23119-crRNA is coated on LB plates containing kanamycin and chloramphenicol, bacterial cells are collected after overnight culture at 37 ℃, the bacterial concentration is adjusted to OD 600.6-0.8, IPTG 0.2mM is added, and induction is performed for 4h at 37 ℃. FastPure EndoFree Plasmid Maxi Kit (vazyme) plasmid extraction was performed to obtain post-subtraction PAM libraries. Primer: PAM-F: GGTCTTCGGTTTCCGTGTT; PAM-R: TGGCGTTGACTCTCAGTCAT. The control samples were obtained by PCR using 30 ng/. Mu.L of plasmid (PAM library) as a template primer, and the experimental samples were obtained by PCR using 30 ng/. Mu.L of plasmid (PAM library after subtraction) as a template. Control samples, experimental samples were submitted to second generation sequencing for data analysis using weblog mapping and Cas-sf1782 (upon in vivo cleavage) identified PAM structure as 5'-NTN-3', where n=a/T/C/G, as shown in fig. 7.
Example 10 application of Cas-s1782 protein in double-stranded nucleic acid editing
This example detects the cis-cleavage activity of double-stranded DNA of Cas-sf1782 in vitro. In this example, the Cas-sf1782 protein is recognized and bound to the target nucleic acid by using the gRNA that can be paired with the target nucleic acid, thereby cleaving the target nucleic acid in the system, and performing agarose electrophoresis detection on the cleaved target nucleic acid.
In this example, the target nucleic acid was selected to be double-stranded DNA (plasmid), T-N-B, which has the sequence:
ligating into Vector T-Vector-pEASY-Blunt Simple Cloning Vector; the italic part is PAM sequence, n=a/T/C/G, the underlined region is the targeting region.
gRNA:Cas-sf1782-nbg1:
CUAACUGUGUGAGCUUCUAACCGAAGCUAAUGACACCCCCCAGCGCUUCAGCGUUC(underlined region is the targeting region)
The following reaction system is adopted: 20 μl system, buffer: tris-HAC 40mM, mg (AC) 2 30mM, BSA 120. Mu.g/ml, DTT 12mM, pH7.0, final Cas-sf1782 concentration of 100nM, final gRNA concentration of 200nM, final double-stranded target nucleic acid concentration of 5 ng/. Mu.L. Incubation at 37℃for 1h and 85℃for 20min. The cleavage products were subjected to agarose electrophoresis to detect Cas-sf1782 cleavage capacity. The experimental group added Cas-sf1782 protein, gRNA and target nucleic acid, and the control group (CK) did not add gRNA.
As a result, as shown in FIG. 8, cas-sf1782 in the experimental group with PAM of 5'-NTT-3', 5'-TTC-3', 5'-CAA-3' was able to cleave double stranded nucleic acid in the system, showing a distinct cleavage band, where N=A/T/C/G, compared to the control without gRNA. This suggests that Cas-sf1782 (when cleaved in vitro) can be used for cleavage and editing of double stranded target nucleic acids with PAM of 5'-NTT-3', 5'-TTC-3', 5 '-CAA-3'.
EXAMPLE 11 PAM identification of Cas-sf2431 protein
Construction of Cas-sf2431 protein expression plasmid: the nucleic acid sequence is subjected to genetic synthesis after being subjected to humanized codon optimization, and is connected with an escherichia coli expression vector PeT28 (a) +vector. The addition of the JM23119 promoter to the vector PeT28 (a) + -Cas-sf2431 initiates transcription of Cas-sf2431 CrRNA. Forming a carrier: peT28 (a) + -Cas-sf2431-JM23119-crRNA, crRNA sequence: GUGUCAGAAGACUUAUAAAAUGAGUCUUAUUGUGACUCCCCUACGUGCUGCUGAAGUUGCUnderlined is the target sequence; construction of PAM library: synthetic sequence CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTCAGCGTTCNNNNNNTCCCCTACGTGCTGCTGAAGTTGCCCGCAA, N is a random deoxynucleotide, underlined is the target sequence. The vector was filled in with Klenow enzyme and ligated into the pacyc184 vector. And (3) converting escherichia coli, and extracting plasmids to form a PAM library.
PAM library subtraction experiment: preparation of competence: BL21 (DE 3) -PeT28 (a) + -Cas-sf2431-JM23119-crRNA. PAM library plasmid transformation competence: BL21 (DE 3) -PeT28 (a) + -Cas-sf2431-JM23119-crRNA was spread on LB plates containing kanamycin and chloramphenicol, cultured overnight at 37℃and then the cells were collected. FastPure EndoFree Plasmid Maxi Kit (vazyme) plasmid extraction was performed to obtain post-subtraction PAM libraries. Primer: PAM-F: GGTCTTCGGTTTCCGTGTT; PAM-R: TGGCGTTGACTCTCAGTCAT. The control samples were obtained by PCR using 30 ng/. Mu.L of plasmid (PAM library) as a template primer, and the experimental samples were obtained by PCR using 30 ng/. Mu.L of plasmid (PAM library after subtraction) as a template. The control and experimental samples were subjected to second generation sequencing for data analysis, using weblog mapping, and Cas-sf2431 identified a PAM structure of 5'-TTC-3', as shown in fig. 9.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.
Claims (18)
1. A Cas protein, characterized in that the Cas protein consists of the amino acid sequence shown in SEQ ID No. 1.
2. A fusion protein comprising the Cas protein of claim 1 and other modifying moieties.
3. An isolated polynucleotide, wherein the polynucleotide is a polynucleotide sequence encoding the Cas protein of claim 1 or a polynucleotide sequence encoding the fusion protein of claim 2.
4. A vector comprising the polynucleotide of claim 3 operably linked to regulatory elements.
5. A CRISPR-Cas system, characterized in that the system comprises the Cas protein of claim 1 and at least one gRNA comprising a cognate repeat sequence capable of binding to the Cas protein of claim 1 and a guide sequence capable of targeting a target sequence, said cognate repeat sequence being as set forth in SEQ ID No. 10.
6. A carrier system comprising one or more carriers, the one or more carriers comprising:
a) A first regulatory element operably linked to a gRNA comprising a cognate repeat sequence capable of binding to the Cas protein of claim 1 and a guide sequence capable of targeting a target sequence, the cognate repeat sequence being as set forth in SEQ ID No. 10;
b) A second regulatory element operably linked to the Cas protein of claim 1 and the Cas protein of claim 1;
wherein components a) and b) are on the same or different supports of the system.
7. A composition, characterized in that it comprises:
(i) A protein component selected from the group consisting of: the Cas protein of claim 1 or the fusion protein of claim 2;
(ii) A nucleic acid component selected from the group consisting of: a gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of the gRNA, or a precursor RNA nucleic acid encoding the gRNA, comprising a cognate repeat sequence capable of binding to the Cas protein of claim 1 and a guide sequence capable of targeting a target sequence, the cognate repeat sequence being as set forth in SEQ ID No. 10;
The protein component and the nucleic acid component are bound to each other to form a complex.
8. An engineered host cell comprising the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7.
9. The Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the use of the composition of claim 7 in gene editing, gene targeting, or gene cleavage, for non-disease diagnosis and therapeutic purposes, the subject of which is exclusive of humans.
10. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, in the preparation of a reagent or kit for gene editing, gene targeting, or gene cleavage.
11. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, in a composition selected from any one or any of the following:
nonspecifically cleaving collateral nucleic acid; detecting nucleic acid;
the application is for non-disease diagnosis and treatment purposes, the subject of the application excluding humans.
12. The use according to claim 11, wherein the non-specifically cleaving lateral branch nucleic acid is a non-specifically cleaving single stranded nucleic acid.
13. A method of editing, targeting, or cleaving a target nucleic acid, the method being a method for non-disease diagnosis and treatment purposes, the method being applied to a subject other than a human, the method comprising contacting the target nucleic acid with the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7.
14. A method of cleaving a single stranded nucleic acid, wherein the method is a method for non-disease diagnosis and treatment purposes, the method being applied to a subject other than a human, the method comprising contacting a population of nucleic acids with the Cas protein of claim 1 and a gRNA comprising a direct sequence capable of binding to the cognate repeat sequence of the Cas protein of claim 1 and capable of targeting a target sequence, the cognate repeat sequence being as set forth in SEQ ID No.10, wherein the population of nucleic acids comprises a target nucleic acid and at least one non-target single stranded nucleic acid, the gRNA is capable of targeting the target nucleic acid, and the Cas protein cleaves the non-target single stranded nucleic acid.
15. A kit for gene editing, gene targeting or gene cleavage, comprising the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7.
16. A kit for detecting a target nucleic acid in a sample, the kit comprising: (a) The Cas protein of claim 1, or a nucleic acid encoding the Cas protein; (b) A gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA comprising the gRNA, or a nucleic acid encoding the precursor RNA, comprising a cognate repeat sequence capable of binding to the Cas protein of claim 1 and a guide sequence capable of targeting a target sequence, the cognate repeat sequence being as set forth in SEQ ID No. 10; and (c) a single-stranded nucleic acid detector that is a single-stranded nucleic acid that does not hybridize to the gRNA.
17. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7 in the preparation of a formulation or kit for which the subject does not include a human, for use:
(i) Editing a target sequence in a target locus to modify an organism;
(ii) Treatment of disease.
18. A method of detecting a target nucleic acid in a sample, the method being a method for non-disease diagnosis and treatment purposes, the method using a subject other than a human, the method comprising contacting the sample with the Cas protein of claim 1, a gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes to the target nucleic acid, and a single-stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein cleaving single-stranded nucleic acid detector, thereby detecting a target nucleic acid; the single stranded nucleic acid detector is a single stranded nucleic acid that does not hybridize to the gRNA.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310952512.7A CN117050973B (en) | 2021-10-29 | 2022-10-28 | Novel Cas enzymes and systems and uses |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111273631 | 2021-10-29 | ||
CN2021112736317 | 2021-10-29 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310952512.7A Division CN117050973B (en) | 2021-10-29 | 2022-10-28 | Novel Cas enzymes and systems and uses |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115851666A CN115851666A (en) | 2023-03-28 |
CN115851666B true CN115851666B (en) | 2023-09-08 |
Family
ID=85661985
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310952512.7A Active CN117050973B (en) | 2021-10-29 | 2022-10-28 | Novel Cas enzymes and systems and uses |
CN202211334825.8A Active CN115851666B (en) | 2021-10-29 | 2022-10-28 | Novel Cas enzymes and systems and uses |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310952512.7A Active CN117050973B (en) | 2021-10-29 | 2022-10-28 | Novel Cas enzymes and systems and uses |
Country Status (1)
Country | Link |
---|---|
CN (2) | CN117050973B (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111690773B (en) * | 2020-06-17 | 2021-08-20 | 山东舜丰生物科技有限公司 | Method and system for detecting target nucleic acid by using novel Cas enzyme |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019206233A1 (en) * | 2018-04-25 | 2019-10-31 | 中国农业大学 | Rna-edited crispr/cas effector protein and system |
JP7460178B2 (en) * | 2018-11-15 | 2024-04-02 | 中国▲農▼▲業▼大学 | CRISPR-Cas12j enzyme and system |
CN113166744A (en) * | 2018-12-14 | 2021-07-23 | 先锋国际良种公司 | Novel CRISPR-CAS system for genome editing |
KR20240036729A (en) * | 2020-03-06 | 2024-03-20 | 메타지노미, 인크. | Class ii, type v crispr systems |
CN113337502B (en) * | 2020-09-25 | 2022-07-08 | 山东舜丰生物科技有限公司 | gRNA and its use |
-
2022
- 2022-10-28 CN CN202310952512.7A patent/CN117050973B/en active Active
- 2022-10-28 CN CN202211334825.8A patent/CN115851666B/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111690773B (en) * | 2020-06-17 | 2021-08-20 | 山东舜丰生物科技有限公司 | Method and system for detecting target nucleic acid by using novel Cas enzyme |
Also Published As
Publication number | Publication date |
---|---|
CN117050973B (en) | 2024-07-19 |
CN117050973A (en) | 2023-11-14 |
CN115851666A (en) | 2023-03-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN115992115B (en) | Novel CRISPR enzymes and systems and uses | |
CN114517190B (en) | CRISPR enzymes and systems and uses | |
CN116334037A (en) | Novel Cas enzymes and systems and uses | |
CN114410609B (en) | Cas protein with improved activity and application thereof | |
WO2023202116A1 (en) | Cas enzyme, system and use | |
CN116004573B (en) | Cas protein with improved editing activity and application thereof | |
CN114277015B (en) | CRISPR enzyme and application | |
CN116555227A (en) | Novel Cas enzyme and application | |
CN115851666B (en) | Novel Cas enzymes and systems and uses | |
CN116555225B (en) | Cas proteins with improved activity and uses thereof | |
CN115261359B (en) | Novel CRISPR enzyme and system and application | |
CN117070498B (en) | Mutant CRISPR-Cas proteins and uses thereof | |
CN116790559B (en) | HNH domain-fused V-type Cas enzyme and application thereof | |
CN115975986B (en) | Mutant Cas12j proteins and uses thereof | |
CN116179510A (en) | Cas enzymes and systems and uses | |
CN117625578A (en) | CRISPR enzyme, system and application | |
CN116218816A (en) | Novel Cas12 enzymes and systems and uses | |
CN116286733A (en) | Cas12b gene editing enzymes and systems and uses | |
CN116083401A (en) | Novel Cas enzymes and systems and uses | |
CN116716277A (en) | Cas enzyme and application thereof | |
CN117247920A (en) | Novel CRISPR enzyme, system and application | |
CN118406671A (en) | Novel Cas enzymes and systems and uses | |
CN116286739A (en) | Mutant Cas proteins and uses thereof | |
CN116200369A (en) | Novel Cas enzyme and application thereof | |
CN117603943A (en) | Cas protein with improved editing efficiency and application 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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |