CN114774459B - 一种香蕉枯萎菌CRISPR/Cas9基因编辑载体、制备方法及应用 - Google Patents
一种香蕉枯萎菌CRISPR/Cas9基因编辑载体、制备方法及应用 Download PDFInfo
- Publication number
- CN114774459B CN114774459B CN202210606127.2A CN202210606127A CN114774459B CN 114774459 B CN114774459 B CN 114774459B CN 202210606127 A CN202210606127 A CN 202210606127A CN 114774459 B CN114774459 B CN 114774459B
- Authority
- CN
- China
- Prior art keywords
- cas9
- sequence
- crispr
- vector
- gene editing
- 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
- 108091033409 CRISPR Proteins 0.000 title claims abstract description 100
- 238000010362 genome editing Methods 0.000 title claims abstract description 71
- 239000013598 vector Substances 0.000 title claims abstract description 60
- 241000223218 Fusarium Species 0.000 title claims abstract description 41
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 241000234295 Musa Species 0.000 title claims abstract description 39
- 235000018290 Musa x paradisiaca Nutrition 0.000 title claims abstract description 39
- 238000010354 CRISPR gene editing Methods 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 230000014509 gene expression Effects 0.000 claims abstract description 33
- 108020005004 Guide RNA Proteins 0.000 claims abstract description 32
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 26
- 108020004705 Codon Proteins 0.000 claims abstract description 22
- 239000013612 plasmid Substances 0.000 claims abstract description 19
- 108010077850 Nuclear Localization Signals Proteins 0.000 claims abstract description 16
- 238000001727 in vivo Methods 0.000 claims abstract description 6
- 208000009869 Neu-Laxova syndrome Diseases 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 32
- 210000001938 protoplast Anatomy 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 15
- 238000012216 screening Methods 0.000 claims description 13
- 238000005457 optimization Methods 0.000 claims description 10
- 238000013461 design Methods 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 claims description 3
- 230000008929 regeneration Effects 0.000 claims description 3
- 238000011069 regeneration method Methods 0.000 claims description 3
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 108700007698 Genetic Terminator Regions Proteins 0.000 claims 1
- 230000000977 initiatory effect Effects 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 9
- 108020004414 DNA Proteins 0.000 description 23
- 238000004458 analytical method Methods 0.000 description 17
- 108700008625 Reporter Genes Proteins 0.000 description 15
- 241000233866 Fungi Species 0.000 description 10
- 101100478440 Gibberella moniliformis SSN8 gene Proteins 0.000 description 10
- 101150050575 URA3 gene Proteins 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000010586 diagram Methods 0.000 description 7
- 101100246753 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) pyrF gene Proteins 0.000 description 6
- 101150065050 carS gene Proteins 0.000 description 6
- 230000004927 fusion Effects 0.000 description 6
- 238000003209 gene knockout Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 230000009466 transformation Effects 0.000 description 5
- 101150083885 BIK1 gene Proteins 0.000 description 4
- 241000223221 Fusarium oxysporum Species 0.000 description 4
- 108010055012 Orotidine-5'-phosphate decarboxylase Proteins 0.000 description 4
- 238000002944 PCR assay Methods 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000006801 homologous recombination Effects 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- 230000007918 pathogenicity Effects 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- LKJPYSCBVHEWIU-KRWDZBQOSA-N (R)-bicalutamide Chemical compound C([C@@](O)(C)C(=O)NC=1C=C(C(C#N)=CC=1)C(F)(F)F)S(=O)(=O)C1=CC=C(F)C=C1 LKJPYSCBVHEWIU-KRWDZBQOSA-N 0.000 description 3
- 102000016736 Cyclin Human genes 0.000 description 3
- 108050006400 Cyclin Proteins 0.000 description 3
- 241000221778 Fusarium fujikuroi Species 0.000 description 3
- 102100037214 Orotidine 5'-phosphate decarboxylase Human genes 0.000 description 3
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 229960000997 bicalutamide Drugs 0.000 description 3
- 235000021466 carotenoid Nutrition 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000012921 fluorescence analysis Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 230000003950 pathogenic mechanism Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 108020005075 5S Ribosomal RNA Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 description 2
- 108010030975 Polyketide Synthases Proteins 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 241000193996 Streptococcus pyogenes Species 0.000 description 2
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
- 150000001747 carotenoids Chemical class 0.000 description 2
- 101150038500 cas9 gene Proteins 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007457 establishment of nucleus localization Effects 0.000 description 2
- 230000034184 interaction with host Effects 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 230000030648 nucleus localization Effects 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000001054 red pigment Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 101150039504 6 gene Proteins 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 102000002428 Cyclin C Human genes 0.000 description 1
- 108010068155 Cyclin C Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- -1 Fusarium carotenoids Chemical class 0.000 description 1
- 241000233732 Fusarium verticillioides Species 0.000 description 1
- 101710103773 Histone H2B Proteins 0.000 description 1
- 102100021639 Histone H2B type 1-K Human genes 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- LTYOQGRJFJAKNA-KKIMTKSISA-N Malonyl CoA Natural products S(C(=O)CC(=O)O)CCNC(=O)CCNC(=O)[C@@H](O)C(CO[P@](=O)(O[P@](=O)(OC[C@H]1[C@@H](OP(=O)(O)O)[C@@H](O)[C@@H](n2c3ncnc(N)c3nc2)O1)O)O)(C)C LTYOQGRJFJAKNA-KKIMTKSISA-N 0.000 description 1
- 150000004008 N-nitroso compounds Chemical class 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 101710118538 Protease Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 101150078331 ama-1 gene Proteins 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000002962 chemical mutagen Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000008380 degradant Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000003008 fumonisin Substances 0.000 description 1
- 238000003197 gene knockdown Methods 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 101150073906 gpdA gene Proteins 0.000 description 1
- 101150095733 gpsA gene Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- LTYOQGRJFJAKNA-DVVLENMVSA-N malonyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CC(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 LTYOQGRJFJAKNA-DVVLENMVSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000005080 plant death Effects 0.000 description 1
- 229930001119 polyketide Natural products 0.000 description 1
- 150000003881 polyketide derivatives Chemical class 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 108091035539 telomere Proteins 0.000 description 1
- 102000055501 telomere Human genes 0.000 description 1
- 210000003411 telomere Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000001018 virulence Effects 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- 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
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供一种香蕉枯萎菌CRISPR/Cas9基因编辑载体、制备方法及应用,该载体包括Cas9和sgRNA表达框;Cas9表达框中,包括PgpdA启动子、优化密码子Foc4‑Cas9和密码子Foc4‑Cas9两端携带的H2BNLS核定位信号;sgRNA表达框中,包括Pol III型5SrRNA启动子和gRNA序列N20;本发明构建的香蕉枯萎菌CRISPR/Cas9基因编辑载体,实现了香蕉枯萎菌Foc4的更高编辑效率的体内表达Cas9和sgRNA的质粒型CRISPR/Cas9基因编辑技术,操作简便且稳定,充分提高香蕉枯萎菌CRISPR/Cas9基因编辑效率。
Description
技术领域
本发明涉及生物技术领域,特别涉及一种香蕉枯萎菌CRISPR/Cas9基因编辑载体、制备方法及应用。
背景技术
丝状真菌(filamentous fungi)广泛存在于自然界不同生境当中,是自然生态系统中的主要成员和有机物质的主要降解者,其生理活动与人类健康、生产生活等都密切相关。随着大量丝状真菌基因组测序及组装的完成,丝状真菌的功能基因组研究与遗传改造等是目前迫切需要解决的问题。丝状真菌作为多细胞的真核微生物,遗传背景较细菌等原核生物复杂,遗传操作困难等因素导致丝状真菌的分子生物学和遗传学研究进展相对缓慢。CRISPR/Cas9基因编辑技术在丝状真菌上的开发和应用,极大促进了真菌的生物学和遗传学的研究,其在丝状真菌功能基因组研究、代谢途径重构、精确表达调控、合成生物学及遗传育种方面具有重要应用前景,但Cas9密码子的选择、启动子的强弱,在宿主内瞬时表达以及组成型或者诱导型的Cas9和sgRNA等因素条件将会对基因组编辑的效率和准确性产生影响。
香蕉枯萎病(Fusarium oxysporum f.sp.cubense,Foc)是破坏香蕉维管束导致植株死亡的毁灭性土传病害,尤其是4号生理小种(Foc race4,Foc4)给香蕉产业和种植者造成巨大经济损失。如何选择快速稳定、操作简便、经济高效的基因编辑方式十分重要,往往大量前期试验和分析对各种因素进行评估分析。由于对于镰刀菌而言,没有曲霉的AMA1自主复制载体无法做到无抗性多循环基因编辑,含有端粒的自主复制载体(pTEL-Fen)是否能够在Fusarium上成功应用尚未实验验证。因此,排除这两个自主复制载体外,Foc4的基因编辑方法只有质粒型和RNP两种方式选择,但质粒型和RNP两种基因编辑都需要筛选标记,因此,构建一种可重复、高效率且稳定的香蕉枯萎菌CRISPR/Cas9基因编辑载体,克服以往基因敲除受限于筛选标记的问题,为解析枯萎菌在与寄主香蕉分子互作中致病机制以及后续采用HIGS技术培育抗枯品系提供理论基础和新的靶标选择。
发明内容
鉴于此,本发明提出一种香蕉枯萎菌CRISPR/Cas9基因编辑载体、制备方法及应用。
本发明的技术方案是这样实现的:
一种香蕉枯萎菌CRISPR/Cas9基因编辑的载体,包括Cas9和sgRNA表达框;所述Cas9表达框中,包括PgpdA启动子、优化密码子Foc4-Cas9和密码子Foc4-Cas9两端携带的H2BNLS核定位信号;所述sgRNA表达框中,包括Pol III型5SrRNA启动子和gRNA序列N20。
进一步说明,所述载体由所述PgpdA启动子启动优化密码子Foc4-Cas9,所述PolIII型5SrRNA启动子启动sgRNA表达。
进一步说明,所述优化密码子Foc4-Cas9的编码基因的核苷酸序列如SEQ ID NO.1所示。
进一步说明,所述NLSH2B核定位信号的核苷酸序列如SEQ ID NO.2所示。
进一步说明,所述gRNA序列N20的核苷酸序列如SEQ ID NO.3-14所示。
一种香蕉枯萎菌CRISPR/Cas9基因编辑载体的构建方法,包括以下步骤:
(1)优化pFocCas9-HTBNLS-hph载体:
设计优化密码子Foc4-Cas9;采用PgpdA启动子启动优化密码子Foc4-Cas9蛋白;并选用H2BNLS作为核定位信号,在优化密码子Foc4-Cas9蛋白的两端携带H2BNLS核定位信号,得到优化后编辑载体pFocCas9-HTBNLS-hph;
(2)选择Pol III型5SrRNA启动子,用于启动sgRNA体内表达;
(3)设计gRNA序列N20,确定gRNA的序列;
(4)依据N20序列overlap PCR方法,构建5SrRNA-sgRNA-T6序列,得到携带N20序列的5SrRNA-N20-sgRNA-T6序列;
(5)将5SrRNA-N20-sgRNA-T6序列导入内切酶EcoR I酶切的编辑载体pFocCas9-HTBNLS-hph,构建得到靶向靶标基因的基因编辑载体。
一种香蕉枯萎菌CRISPR/Cas9基因编辑方法,将上述构建的靶向靶标基因的基因编辑载体、供体质粒dDNA,混入Foc4的原生质体,冰浴30min,再生筛选培养基采用100μg/mL潮霉素HYG抗性,原生质体转化后,筛选得到转化子。
一种上述香蕉枯萎菌CRISPR/Cas9基因编辑载体,在提高香蕉枯萎菌CRISPR/Cas9基因编辑效率中的应用。
一种上述香蕉枯萎菌CRISPR/Cas9基因编辑载体在解析枯萎菌与寄主香蕉分子互作的致病机制中的应用。
与现有技术相比,本发明的有益效果是:本发明构建了香蕉枯萎菌CRISPR/Cas9基因编辑载体,通过Cas9密码子的优化、核定位NLS筛选、sgRNA启动子的选择和gRNA序列N20的设计,实现香蕉枯萎菌Foc4的更高编辑效率的体内表达Cas9和sgRNA的质粒型CRISPR/Cas9基因编辑技术,操作简便且稳定,可以同时敲除2-6个基因,扩大致病相关基因筛选范围,克服了以往基因敲除受限于筛选标记的问题,为解析枯萎菌在与寄主香蕉分子互作中致病机制以及后续采用HIGS技术培育抗枯品系提供理论基础和新的靶标选择,从而有效推进了香蕉枯萎菌的分子遗传学研究。
附图说明
图1为本发明香蕉枯萎菌Foc4 CRISPR/Cas9基因编辑技术体系的核心序列示意图;
图2为发明实施例2H2B进化树分析图;
图3为发明实施例2NLS序列比对分析图;
图4为本发明实施例2NLS融合GFP后的荧光分析图;
图5为本发明5SrRNA表达框以及引物和5SrRNA、sgRNA定量分析图;
图6为本发明Foc4中过表达Cas9后的致病力分析和转化子PDA表型分析图;
图7为本发明基因编辑载体pFocCas9-HTBNLS-hph载体的示意图;
图8为本发明基因编辑载体pFocCas9-HTBNLS-hph载体序列一览图;
图9为本发明以FCC1为内源报告基因的基因编辑、gRNA序列、突变体的表型分析、切割效率分析和PCR检测验结果示意图;
图10为本发明以URA3为内源报告基因的基因编辑、gRNA序列、融合表达GFP的荧光分析和PCR检测验结果示意图;
图11为本发明以BIK1为内源报告基因的基因编辑、gRNA序列、突变体的表型分析和PCR检测验结果示意图;
图12为本发明以carS为内源报告基因的基因编辑、gRNA序列、突变体的表型分析和PCR检测验结果示意图;
图13为本发明以FCC1为靶标对比基因编辑和基因敲除的效率结果示意图。
具体实施方式
为了更好理解本发明技术内容,下面提供具体实施例,对本发明做进一步的说明。
本发明实施例所用的实验方法如无特殊说明,均为常规方法。
本发明实施例所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1-一种香蕉枯萎菌CRISPR/Cas9基因编辑的载体,包括Cas9和sgRNA表达框;
所述Cas9表达框中,包括PgpdA启动子、优化密码子Foc4-Cas9和密码子Foc4-Cas9两端携带的H2BNLS核定位信号;
所述sgRNA表达框中,包括Pol III型5SrRNA启动子和gRNA序列N20。
实施例2-香蕉枯萎菌Foc4 CRISPR/Cas9基因编辑技术的优化
本发明对香蕉枯萎菌CRISPR/Cas9基因编辑技术的优化,主要是pFocCas9-HTBNLS-hph编辑载体中的4个核心序列的优化,其包括两个核心表达框架:Cas9和sgRNA,在第一个Cas9表达框的Foc4-Cas9密码子优化后的序列、核定位NLS序列是决定编辑成功的核心因素;第二个表达框中sgRNA启动子的选择以及N20设计也是核心引物,如图1所示。
(1)Foc4-Cas9序列的密码子优化
合成基因Cas9-Fol 4318bp:密码子优化后的Cas9的编码基因的核苷酸序列如SEQID NO.1所示。
(2)核定位信号NLS的保守性分析与选择
如图2所示的H2B进化树分析以及如图3所示的NLS序列比对分析。通过生物信息分析,组蛋白H2B N端6-45aa具有特异性,NLS序列中端与其他物种高度保守,如图4所示的NLS融合GFP后的荧光分析,通过原生质体转化将H2B融合GFP导入Foc4后,阳性转化子中出现与pCT74不同的荧光信号分布,pCT74携带的GFP在整个细胞都表达,而携带NLSH2B的GFP则集中在细胞核中表达,并且NLSH2B核定位信号还存在剂量效应,双NLSH2B核定位信号的荧光强度比单个的NLSH2B核定位信号好。
NLSH2B核定位信号的核苷酸序列如SEQ ID NO.2所示。
(3)对sgRNA启动子的选择
5SrRNA启动子属于Pol III型启动子,具有U6等Pol II启动子无法比拟的优势。本发明对5SrRNA数据库http://biobases.ibch.poznan.pl/5SData/进行搜索比对,找到Foc45S rRNA启动子序列,构建pFocCas9-HTBNLS-hph基因编辑载体,qPCR分析5SrRNA启动子在Foc4中应用的可行性。
如图5所示(A为5SrRNA表达框以及引物示意图;B为5SrRNA以及sgRNA定量分析),对Foc4,空载体pUC-fFuCas9-HTBNLS-hph,以及靶向FCC1的pFocCas9-HTBNLS-fcc1进行了qPCR表达分析。其中,Foc4中含有5SrRNA启动子序列,不含有sgRNA序列,因此5SrRNA以及sgRNA表达量低;水稻恶苗病菌(F.fujikuroi)的基因编辑空载体pUC-fFuCas9-HTBNLS-hph不含有5SrRNA表达框,只有在设计好N20后才插入到原始载体构建成为靶向靶标基因的编辑载体,与Foc4的检测结果一样,而pFocCas9-HTBNLS-fcc1阳性转化子sgRNA高丰度表达,因此,5SrRNA在Foc4中可以启动sgRNA表达。
(4)验证Cas9过表达对Foc4表型和毒力的影响
本发明分别将含有酿脓链球菌(Streptococcus pyogenes)Cas9的丝状真菌CRISPR基因编辑载体pFC332以及水稻恶苗病菌(F.fujikuroi)的基因编辑载体pUC-fFuCas9-HTBNLS-hph分别通过原生质体方法导入Foc4,得到的阳性转化子分别进行PDA表型和致病力分析,结果显示,Foc4中过表达Cas9并不影响转化子的表型和致病力,如图6所示(A为Foc4中过表达Cas9后的致病力分析;B,Foc4中过表达Cas9后的转化子PDA表型分析)。
(5)Foc4内源报告基因的选择和N20设计
①gRNA序列N20的设计。guide RNA设计网站:Eukaryotic Pathogen CRISPRguide RNA/DNA Design Tool(http://grna.ctegd.uga.edu/)和CRISPOR(http://crispor.tefor.net/),综合比对分析后确定了gRNA的序列,
②为了构建Foc4的CRISPR/Cas9基因编辑技术,本发明筛选四个内源报告基因作为编辑的靶标,用于评估编辑的效率,这四个内源报告基因分别为细胞周期蛋白FCC1、编码乳清苷-5′-磷酸脱羧酶的尿嘧啶缺陷型URA3基因、编码比卡菌素聚酮合酶编码的Bik1基因、以及参与类胡萝卜素次生代谢产物生物合成的carS基因。
实施例3-香蕉枯萎菌CRISPR/Cas9基因编辑载体的优化
根据实施例2香蕉枯萎菌Foc4 CRISPR/Cas9基因编辑技术的优化,包括两个核心表达框架:Cas9和sgRNA。采用Pol III型启动子5S rRNA启动sgRNA体内表达,选用H2BNLS作为核定位信号,在密码子优化后Cas9蛋白两端都携带H2BNLS核定位信号增强Cas9进入细胞核的能力。
如图7和8所示,本发明优化后的基因编辑载体pFocCas9-HTBNLS-hph载体示意图。载体的构建采用PgpdA启动子启动密码子优化的Cas9蛋白,在Cas9两端携带NLS核定位信号;采用Pol III型5SrRNA启动子启动sgRNA表达。
实施例4-靶向靶标基因的基因编辑载体的构建方法
依据N20序列overlap PCR方法构建5SrRNA-sgRNA-T6序列。第一轮PCR方法分别扩增5SrRNA-sgRNA-T6上、下两端序列;第二轮PCR用5SrRNA-U-F-EcoRI/5SrRNA-U-R-EcoRI引物对overlap PCR融合扩增上、下两段序列成为携带N20的5SrRNA-N20-sgRNA-T6序列。
将5SrRNA-N20-sgRNA-T6表达框同源重组导入编辑载体。内切酶EcoR I酶切基因编辑载体pFocCas9-HTBNLS-hph后回收载体片段,利用诺唯赞(Vazyme)IIOne Step Cloning Kit(#C112)重组克隆试剂盒,将5SrRNA-N20-sgRNA-T6序列同源重组导入载体构建成靶向靶标基因的基因编辑载体,比如靶向FCC1的pFocCas9-HTBNLS-fcc1编辑载体,该载体采用体内表达的Cas9和sgRNA质粒进行基因编辑。
实施例5-香蕉枯萎菌CRISPR/Cas9基因编辑
分别取实施例4中构建的5μgpFocCas9-HTBNLS-hph编辑载体、供体质粒dDNA(含有包含靶标基因5’上游1kb左右的同源臂序列、抗性基因序列、3’下游1kb左右的同源臂序列的HDR模板)混入200μl 1×107个/mL的香蕉枯萎菌原生质体,轻轻混匀后冰浴30min,再生筛选培养基采用100μg/mL潮霉素HYG抗性,其他步骤与原生质体转化方法一致(香蕉枯萎菌原生质体制备以及PEG介导的原生质体转化参照Yu et al.(2004)、Hwang&Ahn(2016)以及Wang et al.(2018)的方法)。基因编辑后的转化后筛选阳性转化子的方法与基因敲除阳性转化子筛选方法一致,差别在于基因敲除突变体基因位点被抗性基因HYG取代;而基因编辑后的突变体中基因没有被抗性基因取代位点,而是被重组删除了。
实施例6-基因编辑突变体获得及PCR检测鉴定转化子
将pFocCas9-HTBNLS-hph和dDNA等体积混合后按照上述的原生质体转化方法进行转化。原生质体转化后挑取转化子CTAB法提取DNA后,引物Outside引物对检测靶标基因是否被成功敲除;而Inside引物对内源基因进行检测;Cas9引物对检测编辑突变体中的Cas9蛋白;sgRNA引物对检测转化子中sgRNA的表达(具体引物对序列见表1)。
表1引物序列表
实施例7-内源报告基因验证香蕉枯萎菌CRISPR/Cas9基因编辑
分别采用4个内源报告基因进行原生质体转化、编辑突变体的PCR检测及表型分析
(1)FCC1(FOIG_04434):
细胞周期蛋白C1(Fusarium cyclin C1,FCC1)是2001年在轮枝镰刀菌(Fusariumverticillioides)上首次发现,含有C型细胞周期蛋白,参与次生代代谢产物伏马菌素FB1(fumonisin B1,FB1)的生物合成以及轮枝镰刀菌的孢子形成。通过生物信息学分析软件SMART(Simple Modular Architecture Research Tool)(http://smart.embl.de)分析Foc4菌株含有Cyclin Box功能域的FCC1同源基因(FOIG_04434),Foc4 FCC1敲除缺失突变体出现典型的红色菌落可作为内源报告基因。
如图9所示,(A)为FCC1内源报告基因为靶标开展的基因编辑示意图。FCC1的供体质粒pUC19-FCC1-HDR、FCC1的三个gRNA序列的靶标DNA区间以及引物均标注在上面。(B)为设计的三个gRNA序列以及构建好的编辑载体pFocCas9-HTBNLS-hph-fcc1与供体质粒pUC19-FCC1-HDR共转化原生质体后的基因编辑效率;(C)为ΔFoc4fcc11(HDR)基因编辑后敲除突变体的表型分析,突变体菌落出现典型的红色表型;(D)为以FCC1为靶标基因设计的其他gRNA序列体外Cas9切割效率分析,与实际原生质体转化存在一定差异,可能是gRNA与DNA结合的稳定性存在差异;(E)为ΔFoc4fcc1(HDR)基因敲除突变体的PCR检测验。
(2)URA3(FOIG_03436):
URA3基因编码的乳清苷-5′-磷酸脱羧酶是酵母尿嘧啶核苷酸合成过程中的关键酶,Foc4中也存在URA3(基因编号FOIG_03436)编码乳清苷5-磷酸脱羧酶(Orotidine 5'-phosphate decarboxylase)。
如图10所示,(A)以URA3内源报告基因为靶标开展的基因编辑示意图。URA3的供体质粒pUC19-URA3-HDR、URA3的三个gRNA序列的靶标DNA区间以及引物均标注在上面。(B)设计的三个gRNA序列以及构建好的编辑载体pFocCas9-HTBNLS-hph-URA3与供体质粒pUC19-URA3-HDR共转化原生质体后的基因编辑效率;(C)URA3基因编辑供体质粒中融合表达GFP,因此阳性转化子中出现绿色荧光,方便后续筛选鉴定;(D)ΔFoc4ura3(HDR)基因敲除突变体的PCR检测验。
(3)Bik1(FOIG_14908):
bik1基因编码催化抗生性聚酮化合物合成的关键酶。其中,bik1全长6270nt,含有3个内含子,编码2009个氨基酸。编码I型聚酮合酶,将1个乙酰辅酶A和八个丙二酰辅酶A聚合形成比卡菌素前体物,负责红色色素比卡菌素的生物合成。Bik1具有成为内源报告基因的特征和潜力。Δbik1敲除突变体不能合成比卡菌素从而丧失了红色色素合成能力,摇培后的菌液呈白色,与野生型的红色菌液对比明显,直接通过颜色差异即可判断基因编辑是否成功。
如图11所示,(A)以BIK1内源报告基因为靶标开展的基因编辑示意图。BIK1的供体质粒pUC19-BIK1-HDR、BIK1的三个gRNA序列的靶标DNA区间以及引物均标注在上面。(B)设计的三个gRNA序列以及构建好的编辑载体pFocCas9-HTBNLS-hph-Bik1与供体质粒pUC19-BIK1-HDR共转化原生质体后的基因编辑效率;(C)ΔFoc4Bik11(HDR)基因编辑后敲除突变体的表型分析,突变体摇培后出现白色表型;(D)ΔFoc4Bik1(HDR)基因敲除突变体的PCR检测验。
(4)carS(FOIG_05085):
carS基因通过调控下游car结构基因参与调控Fusarium类胡萝卜素的生物合成。甲基硝基亚硝基胍(N-methyl-N'-nitro-N-nitrosoguanidine,MNNG)是一种N-亚硝基化合物,可直接与DNA作用的化学诱变剂。在藤仓镰孢菌(F.fujikuroi)中利用此化学诱变剂首次获得了类胡萝卜素过量表达的carS突变体,carS突变体菌落呈深橙色,carS具有作为内源报告基因的特点和潜力。
如图12所示,(A)以carS内源报告基因为靶标开展的基因编辑示意图。CARS的供体质粒pYF11-carS-HDR、carS的三个gRNA序列的靶标DNA区间以及引物均标注在上面。(B)设计的三个gRNA序列以及构建好的编辑载体pFocCas9-HTBNLS-hph-carS与供体质粒pUC19-CARS-HDR共转化原生质体后的基因编辑效率;(C)ΔFoc4carS1(HDR)基因编辑后敲除突变体的表型分析,突变体摇培后出现白色表型;(D)ΔFoc4carS(HDR)基因敲除突变体的PCR检测验。
实施例8-CRISPR/Cas9编辑和基因敲除的相关性分析
基因编辑的基本原理是同源重组,供体质粒含有靶标基因5’上游1kb左右的同源臂序列、3’下游1kb左右的同源臂序列,与丝状真菌基因敲除时的同源重组HR具有相同的原理,差别在于基因编辑在双链DNA上产生了DSB断裂,可能促进了HR的发生,提高了HR效率。为验证本次推测,本发明将编辑供体质粒单独转化,与基因编辑的效率进行对比分析,如图13所示,结果证实DSB断裂促进了HR重组,提高了编辑效率。因此,CRISPR/Cas9基因编辑是在重组的基础上利用DSB增加了HR重组效率。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 中国热带农业科学院环境与植物保护研究所
<120> 一种香蕉枯萎菌CRISPR/Cas9基因编辑载体、制备方法及应用
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4318
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gtcgacacta gtatggacaa gaagtacagc atcggcctcg atatcggtac taacagcgtt 60
ggttgggccg ttattaccga cgagtataag gtccccagca agaagttcaa ggtccttggt 120
aacaccgacc gacactctat caagaagaac ctcatcggcg ctctgctttt cgattcagga 180
gagacagccg aggctactag acttaagcga accgctagac gacgatacac tcgacgaaag 240
aaccgcattt gctacctcca ggagatcttc agcaacgaga tggctaaggt cgacgatagc 300
ttcttccaca gacttgagga gagcttcctc gttgaggagg ataagaagca cgagcgacat 360
cctatcttcg gtaacatcgt cgacgaggtt gcttaccacg agaagtaccc taccatctac 420
caccttcgca agaagctcgt cgattctacc gataaggccg atcttcgact catctacctc 480
gctcttgctc atatgatcaa gttccgcggc catttcctca tcgagggtga tcttaacccc 540
gataactcag acgtcgataa gctcttcatc cagctcgttc agacctacaa ccagctcttc 600
gaggagaacc ctatcaacgc ttcaggagtt gacgctaagg ctatcctttc cgctcgactt 660
tctaagtctc gccgacttga gaaccttatc gctcagttgc ctggagagaa gaagaacggt 720
ttgttcggca accttatcgc tctttctctc ggtctcactc ctaacttcaa gagcaacttc 780
gaccttgccg aggacgctaa gcttcagctc tctaaggata cctacgacga cgatctcgat 840
aacctccttg ctcagatcgg agatcagtac gccgatttgt tccttgccgc taagaacctt 900
agcgacgcta ttctcctcag cgatatcctt cgagtcaaca ctgagatcac taaggctcct 960
ctctcagctt ctatgatcaa gcgctacgac gagcatcatc aggatctcac tctcctcaag 1020
gctcttgttc gacaacagct tcccgagaag tacaaggaga tcttcttcga ccagagcaag 1080
aacggttacg ccggttatat tgacggagga gctagtcagg aggagttcta caagttcatc 1140
aagcccatcc tcgagaagat ggacggtaca gaggagcttc tcgttaagct caaccgagag 1200
gatctcctca gaaagcagcg aactttcgac aacggtagca tccctcatca gatccatctt 1260
ggagagcttc acgctatcct cagaagacag gaggacttct accctttcct caaggacaac 1320
cgagagaaga tcgagaagat cctcaccttc cgaatccctt actacgtcgg tcctcttgct 1380
agaggtaact ctcgattcgc ttggatgact cgaaagagcg aggagactat caccccttgg 1440
aacttcgagg aggttgttga caagggagct tcagctcagt ctttcatcga gcgcatgacc 1500
aacttcgaca agaaccttcc taacgagaag gtcctcccta agcattctct cctctacgag 1560
tacttcaccg tctacaacga gctcaccaag gtcaagtacg tcacagaggg tatgcgaaag 1620
ccagctttcc tttcaggcga gcagaagaag gctatcgttg atctcctctt caagaccaac 1680
cgaaaggtta ccgtcaagca gctcaaggag gactacttca agaagatcga gtgcttcgac 1740
agcgttgaga tcagcggagt tgaggatcga ttcaacgctt ctctcggtac ttaccacgat 1800
ctcctcaaga tcatcaagga caaggacttc ctggacaacg aggagaacga ggatatcctt 1860
gaggacatcg tcctcactct cactcttttc gaggacagag agatgatcga ggagcgactt 1920
aagacctacg ctcatctctt cgacgacaag gtcatgaagc agctcaagag acgaagatat 1980
accggttggg gtcgactttc tcgaaagctc atcaacggca tccgagataa gcagagcggt 2040
aagaccatcc ttgacttcct caagagcgac ggtttcgcta accgtaactt catgcagctc 2100
atccacgacg atagcctcac tttcaaggag gacatccaga aggctcaggt ttcaggtcaa 2160
ggagattctc tccacgagca tattgctaac cttgccggtt ctcctgctat caagaagggt 2220
atcctccaaa ccgtcaaggt tgttgacgag ctcgttaagg tcatgggtag acacaagcca 2280
gagaacatcg tcatcgagat ggctagagag aaccagacca ctcaaaaggg ccagaagaac 2340
tctcgagaga gaatgaagcg catcgaggag ggtatcaagg agcttggttc tcagatcctt 2400
aaggagcatc ccgttgagaa cactcagctc cagaacgaga agctctacct ctactacctc 2460
cagaacggta gagacatgta cgtcgatcag gagcttgaca tcaaccgact tagcgattac 2520
gacgtcgatc acatcgttcc tcagtctttc ctcaaggacg actctatcga caacaaggtc 2580
ctcactcgaa gcgacaagaa ccgaggtaag agcgataacg tcccttcaga ggaggtcgtc 2640
aagaagatga agaactattg gcgccagctc cttaacgcta agcttatcac ccagcgcaag 2700
ttcgacaacc ttactaaggc cgagagagga ggtctttcag agcttgataa ggccggcttc 2760
atcaagagac agcttgtcga gactcgacag atcactaagc acgttgctca gatcctcgac 2820
tctcgaatga acaccaagta cgacgagaac gacaagctca tcagagaggt caaggtcatc 2880
accctcaagt ctaagctcgt tagcgacttc cgcaaggatt tccagttcta caaggtccgc 2940
gagatcaaca actaccatca cgctcacgac gcttacctta acgccgttgt tggtaccgct 3000
cttatcaaga agtaccccaa gcttgagagc gagttcgttt acggcgatta caaggtctac 3060
gacgtcagaa agatgatcgc caagagcgag caagagattg gtaaggctac cgccaagtac 3120
ttcttctaca gcaacatcat gaacttcttc aagaccgaga tcactcttgc taacggagag 3180
atccgaaagc gacctcttat cgagactaac ggagagacag gagagatcgt ttgggataag 3240
ggtcgagact tcgctaccgt tagaaaggtc ctctctatgc ctcaggtcaa catcgtcaag 3300
aagaccgagg ttcagacagg cggtttctct aaggagtcta tcctccctaa gcgaaacagc 3360
gacaagctta tcgctcgcaa gaaggattgg gaccctaaga agtacggagg tttcgattct 3420
cctaccgttg cttactctgt cctcgttgtt gctaaggtcg agaagggtaa gagcaagaag 3480
ctcaagagcg tcaaggagct tctcggtatc accatcatgg agcgatctag cttcgagaag 3540
aaccctatcg acttcctcga ggctaagggt tacaaggagg tcaagaagga cctcatcatc 3600
aagctcccca agtactctct cttcgagctt gagaacggca gaaagcgaat gcttgcttca 3660
gccggagagc ttcaaaaggg taacgagttg gctctccctt ctaagtacgt caacttcctc 3720
tacctcgcta gccattacga gaagctcaag ggttctccag aggataacga gcagaagcag 3780
ctcttcgttg agcagcataa gcactacctc gacgagatca tcgagcagat cagcgagttc 3840
tctaagcgag ttatccttgc cgacgctaac cttgacaagg ttcttagcgc ctacaacaag 3900
caccgagata agcctatcag agagcaagcc gagaacatca tccacctctt cactcttact 3960
aaccttggcg ctcctgccgc tttcaagtat ttcgacacca ctatcgaccg caagcgatac 4020
acttctacca aggaggttct cgacgctact cttatccacc aaagcatcac cggtctttac 4080
gagactcgaa tcgatctttc tcagcttggc ggagatgtat accctatggc tcccaaggct 4140
gctgacaaga agcccgcctc caaggctccc gctactgcct ccaaggctcc tgagaagaag 4200
gatgctggca agaagactgc cgcctctggt gacaagaaga agcgctccaa gacccgcaag 4260
gagacctact cttcttacat ctacaagtaa tacgtacact taacgttact gaaatcat 4318
<210> 2
<211> 162
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggctccca aggctgctga caagaagccc gcctccaagg ctcccgctac tgcctccaag 60
gctcctgaga agaaggatgc tggcaagaag actgccgcct ctggtgacaa gaagaagcgc 120
tccaagaccc gcaaggagac ctactcttct tacatctaca ag 162
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atatcgcggt cgctatgacg 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tttgcaggct aggtatatcg 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
acagaggcgc gacagatgtg 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctctacttcg ccgacaagat 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gtgggccaga gatgtaaccg 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tgttggacgc ggtatcatca 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agagagaaaa tcgattcccg 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atctttcgcg cagctgacag 20
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gctggccagt gggatcgtag 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
agagagaaaa tcgattcccg 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atctttcgcg cagctgacag 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gctggccagt gggatcgtag 20
Claims (4)
1.一种香蕉枯萎菌CRISPR/Cas9基因编辑的载体,其特征在于:包括Cas9表达框和sgRNA表达框;
所述Cas9表达框中,包括PgpdA启动子和两端携带H2BNLS核定位信号的密码子优化的Foc4-Cas9;所述两端携带H2BNLS核定位信号的密码子优化的Foc4-Cas9的编码基因的核苷酸序列由SEQ ID NO.2所示的序列和SEQ ID NO.1所示的序列,按5’至3’的顺序相连获得;
所述sgRNA表达框中,包括Pol III型5SrRNA启动子和gRNA序列N20,所述gRNA序列N20的核苷酸序列选自SEQ ID NO.12-14所示的序列;
所述载体由所述PgpdA启动子启动Foc4-Cas9,所述Pol III型5SrRNA启动子启动gRNA表达。
2.如权利要求1所述的一种香蕉枯萎菌CRISPR/Cas9基因编辑载体的构建方法,其特征在于:包括以下步骤:
(1)优化pFocCas9-HTBNLS-hph载体:
设计优化密码子Foc4-Cas9;采用PgpdA启动子启动优化密码子Foc4-Cas9蛋白;并选用H2BNLS作为核定位信号,在优化密码子Foc4-Cas9蛋白的两端携带H2BNLS核定位信号,得到优化后编辑载体pFocCas9-HTBNLS-hph;
(2)选择Pol III型5SrRNA启动子,用于启动gRNA体内表达;
(3)设计gRNA序列N20,确定gRNA的序列;
(4)依据N20序列overlap PCR方法,构建5SrRNA-sgRNA-T6序列,得到由Pol III型5SrRNA启动子启动和T6终止子序列终止N20表达的5SrRNA-N20-sgRNA-T6序列;
(5)将5SrRNA-N20-sgRNA-T6序列导入内切酶EcoR I酶切的编辑载体pFocCas9-HTBNLS-hph,构建得到靶向靶标基因的基因编辑载体。
3.一种香蕉枯萎菌CRISPR/Cas9基因编辑方法,其特征在于:将如权利要求2中构建的靶向靶标基因的基因编辑载体和供体质粒dDNA,混入香蕉枯萎病菌4号生理小种Foc4的原生质体,冰浴30min,再生筛选培养基采用100 µg/mL潮霉素HYG抗性,原生质体转化后,筛选得到转化子。
4.一种如权利要求1~3中任意一项所述的香蕉枯萎菌CRISPR/Cas9基因编辑载体在提高香蕉枯萎菌CRISPR/Cas9基因编辑效率中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210606127.2A CN114774459B (zh) | 2022-05-31 | 2022-05-31 | 一种香蕉枯萎菌CRISPR/Cas9基因编辑载体、制备方法及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210606127.2A CN114774459B (zh) | 2022-05-31 | 2022-05-31 | 一种香蕉枯萎菌CRISPR/Cas9基因编辑载体、制备方法及应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114774459A CN114774459A (zh) | 2022-07-22 |
CN114774459B true CN114774459B (zh) | 2024-03-12 |
Family
ID=82421829
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210606127.2A Active CN114774459B (zh) | 2022-05-31 | 2022-05-31 | 一种香蕉枯萎菌CRISPR/Cas9基因编辑载体、制备方法及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114774459B (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107164375A (zh) * | 2017-05-25 | 2017-09-15 | 中国科学院天津工业生物技术研究所 | 一种新型向导RNA表达盒及在CRISPR/Cas系统中的应用 |
CN112553238A (zh) * | 2020-12-10 | 2021-03-26 | 广东省微生物研究所(广东省微生物分析检测中心) | 一种适用于拟盾壳霉FS482的CRISPR/Cas9载体及其构建方法和应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170088845A1 (en) * | 2014-03-14 | 2017-03-30 | The Regents Of The University Of California | Vectors and methods for fungal genome engineering by crispr-cas9 |
-
2022
- 2022-05-31 CN CN202210606127.2A patent/CN114774459B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107164375A (zh) * | 2017-05-25 | 2017-09-15 | 中国科学院天津工业生物技术研究所 | 一种新型向导RNA表达盒及在CRISPR/Cas系统中的应用 |
CN112553238A (zh) * | 2020-12-10 | 2021-03-26 | 广东省微生物研究所(广东省微生物分析检测中心) | 一种适用于拟盾壳霉FS482的CRISPR/Cas9载体及其构建方法和应用 |
Non-Patent Citations (4)
Title |
---|
Controlled Transcription of Regulator Gene carS by Tet-on or by a Strong Promoter Confirms Its Role as a Repressor of Carotenoid Biosynthesis in Fusarium fujikuroi;Julia Marente et al.;《Microorganisms》;第9卷(第1期);摘要 * |
CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Fusarium fujikuroi and Its Application in Strain Engineering for Gibberellic Acid Production;Tian-Qiong Shi et al.;《ACS Synth Biol》;第8卷(第2期);第447页左栏第3段,第452页左栏第2段,补充数据 * |
Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein complexes;Qiang Wang et al.;《Fungal Genet Biol》;第117卷;摘要,第7页3.2节 * |
香蕉枯萎病菌比卡菌素聚酮合酶编码基因Bik1 的鉴定以及作为内源报告基因在CRISPR/Cas9体系中的可行性评估;王艳玮等;《热带作物学报》;摘要,第6页第1.2.7节,第4页1.2.3节,第11页第2.6节,图6 * |
Also Published As
Publication number | Publication date |
---|---|
CN114774459A (zh) | 2022-07-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Schwartz et al. | CRISPRi repression of nonhomologous end‐joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica | |
Rajkumar et al. | Biological parts for Kluyveromyces marxianus synthetic biology | |
Schwartz et al. | Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica | |
Cui et al. | Homology‐independent genome integration enables rapid library construction for enzyme expression and pathway optimization in Yarrowia lipolytica | |
US20170088845A1 (en) | Vectors and methods for fungal genome engineering by crispr-cas9 | |
KR20200026878A (ko) | 균류 균주를 개량하기 위한 htp 게놈 공학 플랫폼 | |
Naseri et al. | COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors | |
US20190144852A1 (en) | Combinatorial Metabolic Engineering Using a CRISPR System | |
US11643648B2 (en) | Method for constructing chimeric plasmid library | |
Jørgensen et al. | A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina) | |
Ciurkot et al. | Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a | |
Crook et al. | Identification of gene knockdown targets conferring enhanced isobutanol and 1-butanol tolerance to Saccharomyces cerevisiae using a tunable RNAi screening approach | |
García-García et al. | Using continuous directed evolution to improve enzymes for plant applications | |
Südfeld et al. | The nucleolus as a genomic safe harbor for strong gene expression in Nannochloropsis oceanica | |
Strucko et al. | CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast | |
Woodcraft et al. | The expanding CRISPR toolbox for natural product discovery and engineering in filamentous fungi | |
WO2019214347A1 (zh) | 一种链霉菌生物合成基因簇负调控因子的遗传筛选方法 | |
CN112481309A (zh) | Ago蛋白的用途及组合物和基因编辑方法 | |
Heo et al. | Simultaneous integration of multiple genes into the Kluyveromyces marxianus chromosome | |
Nora et al. | Synthetic and minimalist vectors for Agrobacterium tumefaciens-mediated transformation of fungi | |
CN114774459B (zh) | 一种香蕉枯萎菌CRISPR/Cas9基因编辑载体、制备方法及应用 | |
US9309542B2 (en) | Recombinant Caldicellulosiruptor bescii and methods of use | |
Fenster et al. | High-throughput functional genomics for energy production | |
Li et al. | Strategy for efficient cloning of biosynthetic gene clusters from fungi | |
Lale et al. | A universal approach to gene expression engineering |
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 |