WO2019196717A1 - Randomly mutated gene editing system and use thereof - Google Patents

Randomly mutated gene editing system and use thereof Download PDF

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WO2019196717A1
WO2019196717A1 PCT/CN2019/081184 CN2019081184W WO2019196717A1 WO 2019196717 A1 WO2019196717 A1 WO 2019196717A1 CN 2019081184 W CN2019081184 W CN 2019081184W WO 2019196717 A1 WO2019196717 A1 WO 2019196717A1
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gene editing
gene
editing system
sgrna1
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姜临建
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青岛清原化合物有限公司
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90Stable introduction of foreign DNA into chromosome
    • C12N15/902Stable introduction of foreign DNA into chromosome using homologous recombination
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  • the invention belongs to the field of biotechnology, and particularly relates to a random mutation gene editing system and an application thereof.
  • Gene editing technology especially CRISPR/Cas9 technology, enables precise gene editing in biological cells.
  • the technical principle is: through a piece of guide RNA (sgRNA or gRNA) combined with endonuclease (such as Cas9, Cpf1, etc.) to form a complex of RNA and protein (referred to as RNP), the complex can search and guide RNA on the genome
  • the complementary target sequence is such that the endonuclease precisely cleaves the bound DNA in this region.
  • the results of the cleavage are diverse and can be double-stranded DNA cleavage (DSB) at the blunt end or sticky end, or single-stranded DNA cleavage (Nick).
  • the repair of the DSB or Nick by the organism's own cells leads to the insertion or deletion of the base (Indel), resulting in a frameshift mutation, resulting in loss of gene function, thereby enabling precise editing of the target gene.
  • the DNA fragment may be repaired and integrated into the DSB or Nick region to achieve precise DNA fragment insertion or replacement.
  • mutant libraries Since many organisms already have clear genomic information, gene editing technology enables large-scale gene knockout of organisms at the genome level to create new mutant libraries.
  • the technical route is to design and synthesize tens of thousands of guide RNAs for different genes, and transfer them to target biological cells by means of viruses, gene guns, and Agrobacterium to obtain mutant libraries. Using this technical route, mutant libraries were successfully constructed on bacteria, animal cells and plants.
  • the need to synthesize tens of thousands of boot RNAs is a major drawback of this technology system.
  • the construction of mutant libraries requires high-throughput transformation systems. Therefore, a technique capable of generating random guide RNA in biological cells will greatly overcome these drawbacks.
  • a first object of the present invention is to provide a gene editing system for random mutation.
  • a randomly mutated gene editing system comprising a targeting element and an editing element, the targeting element directing an editing element to edit a gene, the targeting element being capable of random mutation to direct an editing element pair The new target sequence is edited.
  • the targeting element comprises a combination of one or both of protein, RNA, DNA, or the targeting element comprises a different protein, a different RNA or a different DNA; the editing element It is an enzyme that uses DNA or a base as a substrate.
  • the targeting element can be realized by specific binding of protein to DNA, or by specific binding between nucleic acids (between DNA and DNA, between RNA and DNA, between RNA and RNA).
  • the editing element may be an endonuclease that directly cleaves DNA, or may be a base-based enzyme such as cytosine deaminase or adenine deaminase fused to these endonucleases or Compound.
  • the editing element is an endonuclease
  • the targeting element is two sgRNA sequences: sgRNA1 and sgRNA2, the partial sequence of the sgRNA2 and the sequence of the corresponding sgRNA1 are highly coincident, and the sgRNA1 is guided into the nucleic acid.
  • the dicer is genetically edited in the DNA region where sgRNA2 is expressed, thereby randomly generating a new sgRNA, which can direct the endonuclease for gene editing, thereby generating a random mutation.
  • the above gene editing system includes a CRISPR system.
  • the two sgRNA sequences respectively comprise one or two identical or different sequences.
  • the partial sequence of the sgRNA2 and the sequence of the corresponding sgRNA1 are highly coincident means that the gene sequence expressing sgRNA2 can be recognized by sgRNA1 and cleaved by an endonuclease.
  • the sequence of a certain sgRNA2 comprises the sequence of the corresponding sgRNA1 and an endonuclease recognition site (eg, a PAM sequence), ie, the sgRNA2 partial sequence and the target sequence of sgRNA1 are coincident, and the target of sgRNA1
  • an endonuclease recognition site eg, a PAM sequence
  • the endonuclease recognition site is set, and the endonuclease cleaves the sgRNA2 gene, and then repairs, randomly generates a new sgRNA gene, and expresses a new sgRNA.
  • the endonuclease is a Cas protein or a Cpf1 protein.
  • the Cas protein is Cas9, Cas9n, dCas9 or xCas9.
  • a second object of the present invention is to provide a nucleic acid expressing the above gene editing system.
  • a third object of the present invention is to provide a vector expressing the above gene editing system.
  • the carrier is a single carrier or a dual carrier
  • the single vector refers to a nucleic acid expressing the above-described gene editing system on a vector
  • the double vector refers to a nucleic acid expressing the above-described gene editing system on two vectors, for example, in one embodiment, an endonuclease and The expression genes of the two sgRNA sequences are located on two vectors, respectively.
  • the above vector is a vector capable of expressing the CRISPR system, and may be a plant vector, an animal vector, a bacterial vector or a fungal vector;
  • the above vector is an optional pHEE401E vector plasmid, an adenovirus vector, a pCRISPR vector, a pDONR207 vector, a lenti CRISPR carrier, a pCMV vector, a pU6 vector, a T1706, a pLko vector, and the like.
  • a fourth object of the present invention is to provide a method for random mutation in a cell comprising the steps of introducing the above nucleic acid or the above vector into a host cell.
  • a fifth object of the present invention is to provide a method for constructing a mutant library comprising the steps of introducing the above nucleic acid or the above vector into a host cell.
  • the host is a bacterium, a fungus, a plant or an animal.
  • the host cell is a germ cell in a sexual reproductive organism or a cleavable cell in an asexual reproductive organism and grows into a cell of the individual.
  • the method further comprises: cultivating a next generation cell or an individual of the host cell;
  • nucleic acid or the above vector is repeatedly introduced into the next generation cell or individual of the host as described above.
  • the nucleic acid or the vector is introduced into a plant egg cell, and Cas9 is driven by an egg cell-specific promoter (or other germ cell-specific expression promoter) to sgRNA2-directed constitutive expression of sgRNA.
  • the random mutations generated by the endonuclease editing of the sgRNA1 gene will only occur in the egg cells, and the new sgRNA generated randomly will also edit the plant genome only in the egg cells, and the harvested seeds will have different editing events.
  • a plant can produce tens of thousands of seeds. In theory, most seeds will contain new sgRNA genes. If the genome has matching sequences, new mutations will be produced.
  • plant is understood to mean any differentiated multicellular organism capable of photosynthesis, in particular monocotyledonous or dicotyledonous plants, for example: (1) a food crop: Oryza spp., such as rice. (Oryza sativa), Oryza latifolia, Oryza sativa, Oryza glaberrima, Triticum spp., such as Triticum aestivum, Durum wheat (T.Turgidumssp) .durum); Hordeum spp., such as Hordeum vulgare, Hordeum arizonicum; Secale cereale; Avena spp., such as oat (Avena sativa), wild oats (Avena fatua), Avena byzantina, Avena fatua var.sativa, Avena hybrida, Echinochloa spp., for example, Pennisetum glaucum, sorghum (
  • Averrhoa carambola Actinidia spp., almond (Pr) Unus amygdalus), Musa spp. (banana), Persea spp. (Persea americana), guava (Psidium guajava), Manmie americana, Mangifera Indica), olive (Oleaeuropaea), papaya (Caricapapaya), coconut (Cocos nucifera), Malpighia emarginata, Manilkara zapota, Ananas comosus, Annona Spp.), citrus tree (Citrus spp.), Artocarpus spp., litchi chinensis, Ribes spp., Rubus spp.
  • root crop cassava Genihot spp., Ipomoea batatas, Colocasia esculenta, mustard, onion, alfalfa, oil sedge, yam; (7) vegetable crops: spinacia spp., genus (Phaseolus) Spp.), lettuce (Lactuca sativa), Momordica spp. ), Pestelinum crispum, Capsicum spp., Solanum spp.
  • Lycopersicon Spp. eg, tomatoes (Lycopersicon esculentum), tomatoes (Lycopersicon lycopersicum, Lycopersicon pyriforme), genus Macrotyloma spp.
  • Pistacia vera Anacardium occidentale, macadamia (Macadamia integrifolia), pecans, macadamia, pistachios, almonds, and nuts-producing plants; (17) Other: Arabidopsis, arm grass, valerian, large Foxtail, goosegrass, Cadaba farinosa, algae, Carex elata, ornamental plants, Carissa macrocarpa, Cynara spp., Daucus carota, Dioscorea Spp.), Erianthus sp., Festuca arundinacea, Hemerocallis fulva, Lotus spp., Luzula sylvatica, Medicago sativa, hibiscus Genus (Melilotus spp.), Morus nigra, Nicotiana spp., Olea spp., Ornithopus spp., Pastinaca sativa, elderberry Genus (Sambucus
  • the present invention provides a novel random mutation gene editing system which utilizes the CRISPR system to simultaneously edit the characteristics of a plurality of different sites, and designs two different sgRNAs, one of which is sgRNA sequence and expression.
  • the DNA region of another sgRNA is highly coincident, which results in the sgRNA of the former directing the endonuclease to edit the DNA region expressing the latter sgRNA, thereby randomly generating a new random sgRNA.
  • These new randomly generated sgRNAs once matched to genomic DNA, direct the endonuclease to mutate at these random sites.
  • random mutations can be made in cells, a novel method of random mutation.
  • Mutations can be randomly performed in somatic cells using the gene editing system of the present invention, and various mutations can be performed to varying degrees. If an endogenous enzyme is driven by an egg cell-specific promoter, a large number of fertilized eggs or zygotes with independent mutation events can be obtained, and the process can be repeated in the next generation, but only one or several different sgRNA gene editing systems can be constructed. The tens of thousands of random mutant libraries do not need to design thousands of sgRNAs. This method is efficient and low-cost, and can be used to construct mutant libraries.
  • Figure 1 shows the sgRNA1-1 and sgRNA2-1 expression cassette sequences
  • Figure 2 shows the structure of the expression cassette of the final vector
  • Figure 3 shows the type of sgRNA1-1 region change
  • Figure 4 shows the emergence of albino seedlings in the T2 generation
  • FIG. 5 shows that the PDS3 genes of four albino seedlings have undergone frameshift mutations.
  • Example 1 Design and construction capable of spontaneous targeting to generate new sgRNA target sequences
  • sgRNA2-1 Two expression cassettes expressing sgRNA1-1 and sgRNA2-1, respectively, were constructed by direct gene synthesis.
  • sgRNA2-1 will direct Cas9 to edit the region of DNA that expresses sgRNA1-1, resulting in a new sgRNA sequence.
  • the sgRNA1-1 expression cassette sequence SEQ ID NO: 1 and the sgRNA2-1 expression cassette sequence SEQ ID NO: 2 are shown in FIG.
  • the bacterial solution was applied to YEP solid medium (containing kanamycin and gentamicin) by scribing, in a dark environment at 28 ° C. After 36-48 hours of culture, a single colony was picked and inoculated into 1 ml of liquid YEB medium to which kanamycin and gentamicin had been added, and cultured overnight with shaking (28 ° C, 200 rpm).
  • colony PCR was performed, and the positive clones were selected, added to a 50 ml Erlenmeyer flask, and 25 ml of YEP liquid medium (containing kanamycin and gentamicin) was added, and cultured at 28 ° C and 200 rpm. Until the OD600 value rises to within 0.8-1.0. The supernatant was removed by centrifugation, and the cells were resuspended in the same volume of 5% sucrose solution (100 ml sterile deionized water plus 5 g sucrose), and Silwet L-77 (0.02%) was added to the bacterial solution to mix. .
  • 5% sucrose solution 100 ml sterile deionized water plus 5 g sucrose
  • Silwet L-77 0.02%
  • the appropriate Arabidopsis inflorescence was taken in the bacterial liquid for 0.5-1 min, and the black opaque plastic bag was covered on the Arabidopsis seedlings to create dark conditions.
  • the cells were placed in a greenhouse at 22 ° C for 24 h and then transferred to light culture. Seeds are harvested after the seeds are ripe.
  • Example 3 Screening of transgenic Arabidopsis plants ⁇ Methods and materials used in this section, refer to (3) Chen Y, Wang Z, Ni H, Xu Y, Chen Q*, Jiang L.*2017.CRISPR/Cas9-mediated base -editing system efficiently generates gain-of-function mutations in Arabidopsis.Science China Life Sciences 60,520-523. ⁇
  • the harvested Arabidopsis seeds were sterilized and screened for transgenic plants on MS medium containing hygromycin (25 mg/L).
  • the DNA of the T1 plant was extracted, and the regions of sgRNA1-1 and sgRNA2-1 were subjected to PCR amplification and sequencing. T1 generation plants were selected without editing and T2 seeds were harvested.
  • the DNA of the T2 generation green seedlings was extracted, and the sgRNA1-1 and sgRNA2-1 regions were subjected to PCR amplification and sequencing. As a result, it was found that the sgRNA2-1 region was unchanged, and the sgRNA1-1 region was changed from the original CCTGACCGCCGAACCATGGC to a plurality of different types, thereby producing a new sgRNA, as shown in FIG.
  • T2 seeds were screened on MS plates containing hygromycin, and a large number of albino seedlings appeared, as shown in Table 1 and Figure 4.
  • sgRNA gene editing systems can be designed; or two sgRNA sequences of an sgRNA gene editing system include one or two identical Or different sequences, that is, sgRNA may be three or more, such as sgRNA1 including sgRNA1-1, sgRNA1-2, and the like, and sgRNA2 includes sgRNA2-1, sgRNA2-2, and the like.
  • sgRNA may be three or more, such as sgRNA1 including sgRNA1-1, sgRNA1-2, and the like, and sgRNA2 includes sgRNA2-1, sgRNA2-2, and the like.
  • the technical system of the present invention is applied to a model plant such as Brachypodium serrata, or other aforementioned plants, such as food crops, legume crops, oil crops, fiber crops, fruit crops, root crops, vegetables.
  • Crops, flower crops, medicinal crops, raw crops, pasture crops, sugar crops, beverage crops, turf plants, tree crops, nut crops, etc. also produce corresponding random mutations at specific screening pressures (herbicides, drought) Under abiotic stress or biotic stress such as pests and diseases, it is expected to screen out resistant mutants, and it can also be applied to various animals, so it has good industrial value.

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Abstract

Disclosed in the present application are a gene editing system for randomly mutating genes and the use thereof. The gene editing system of the present invention comprises a targeting element and an editing element, the targeting element directing the editing element to edit a gene, characterized in that the targeting element can be randomly mutated to direct the editing element to edit a new target sequence. By using the system of the present invention, random mutation can be performed in cells, which is a new method for random mutation, and a high-throughput library of thousands of random mutants can be constructed with high efficiency and at low cost.

Description

一种随机突变的基因编辑系统及其应用A random mutation gene editing system and its application 技术领域Technical field
本发明属于生物技术领域,具体涉及一种随机突变的基因编辑系统及其应用。The invention belongs to the field of biotechnology, and particularly relates to a random mutation gene editing system and an application thereof.
背景技术Background technique
基因编辑技术,特别是CRISPR/Cas9技术,在生物细胞中实现了精准的基因编辑。其技术原理是:通过一段引导RNA(sgRNA或gRNA)与DNA内切酶(如Cas9、Cpf1等)结合形成RNA和蛋白的复合物(简称RNP),该复合物可以在基因组上搜索与引导RNA上互补的目标序列,从而使得DNA内切酶精准地在该区域对结合的DNA进行剪切。根据不同的DNA内切酶特性,剪切的结果多种多样,可以是平末端或粘性末端的双链DNA断裂(DSB),也可以是单链DNA断裂(Nick)。生物体的细胞自身对DSB或Nick的修复会导致碱基的插入或删除(Indel),造成移码突变,致使基因功能丧失,从而实现了对目的基因的精准编辑。此外,如果提供DNA修复模板,该DNA片段就可能被修复整合入DSB或Nick区域,从而实现精准的DNA片段插入或替换。Gene editing technology, especially CRISPR/Cas9 technology, enables precise gene editing in biological cells. The technical principle is: through a piece of guide RNA (sgRNA or gRNA) combined with endonuclease (such as Cas9, Cpf1, etc.) to form a complex of RNA and protein (referred to as RNP), the complex can search and guide RNA on the genome The complementary target sequence is such that the endonuclease precisely cleaves the bound DNA in this region. Depending on the nature of the endonuclease, the results of the cleavage are diverse and can be double-stranded DNA cleavage (DSB) at the blunt end or sticky end, or single-stranded DNA cleavage (Nick). The repair of the DSB or Nick by the organism's own cells leads to the insertion or deletion of the base (Indel), resulting in a frameshift mutation, resulting in loss of gene function, thereby enabling precise editing of the target gene. In addition, if a DNA repair template is provided, the DNA fragment may be repaired and integrated into the DSB or Nick region to achieve precise DNA fragment insertion or replacement.
由于很多生物已经具有明确的基因组信息,利用基因编辑技术,能够在基因组水平上对生物进行大规模基因敲除,从而建立新的突变体库。其技术路线是,针对不同的基因设计并合成数以万计的引导RNA,利用病毒、基因枪、农杆菌等手段将其转入到目标生物细胞中,来获得突变体库。利用该技术路线,在细菌、动物细胞和植物上均成功构建了突变体库。但是,需要合成数以万计的引导RNA是该技术体系的一大弊端。此外,要构建突变体库需要高通量的转化体系。因此,一种能够在生物细胞中产生随机引导RNA的技术,将极大克服这些弊端。Since many organisms already have clear genomic information, gene editing technology enables large-scale gene knockout of organisms at the genome level to create new mutant libraries. The technical route is to design and synthesize tens of thousands of guide RNAs for different genes, and transfer them to target biological cells by means of viruses, gene guns, and Agrobacterium to obtain mutant libraries. Using this technical route, mutant libraries were successfully constructed on bacteria, animal cells and plants. However, the need to synthesize tens of thousands of boot RNAs is a major drawback of this technology system. In addition, the construction of mutant libraries requires high-throughput transformation systems. Therefore, a technique capable of generating random guide RNA in biological cells will greatly overcome these drawbacks.
发明内容Summary of the invention
针对上述问题,本发明的第一目的在于提供一种随机突变的基因编辑系统。In view of the above problems, a first object of the present invention is to provide a gene editing system for random mutation.
一种随机突变的基因编辑系统,所述基因编辑系统包括靶向元件和编辑元件,所述靶向元件引导编辑元件对基因进行编辑,所述靶向元件可进行随机突变,从而引导编辑元件对新的靶点序列进行编辑。A randomly mutated gene editing system comprising a targeting element and an editing element, the targeting element directing an editing element to edit a gene, the targeting element being capable of random mutation to direct an editing element pair The new target sequence is edited.
上述基因编辑系统中,所述靶向元件包括蛋白质、RNA、DNA中的一种或两种的结合,或者所述靶向元件包括不同的蛋白质、不同的RNA或不同的DNA;所述编辑元件为以DNA或碱基为底物的酶。In the above gene editing system, the targeting element comprises a combination of one or both of protein, RNA, DNA, or the targeting element comprises a different protein, a different RNA or a different DNA; the editing element It is an enzyme that uses DNA or a base as a substrate.
所述靶向元件可以利用蛋白与DNA的特异性结合来实现,也可以是通过核酸之间的特 异性结合(DNA与DNA之间,RNA与DNA之间的,RNA与RNA之间)来实现。所述编辑元件,可以是直接对DNA进行切割的内切酶,也可以是融合在这些内切酶之上的胞嘧啶脱氨酶、腺嘌呤脱氨酶等以碱基为底物的酶或者化合物。The targeting element can be realized by specific binding of protein to DNA, or by specific binding between nucleic acids (between DNA and DNA, between RNA and DNA, between RNA and RNA). . The editing element may be an endonuclease that directly cleaves DNA, or may be a base-based enzyme such as cytosine deaminase or adenine deaminase fused to these endonucleases or Compound.
上述基因编辑系统中,所述编辑元件为核酸内切酶;所述靶向元件为两种sgRNA序列:sgRNA1和sgRNA2,所述sgRNA2的部分序列和相应sgRNA1的序列高度重合,使sgRNA1引导核酸内切酶在表达sgRNA2的DNA区域进行基因编辑,从而随机产生新的sgRNA,所述新的sgRNA可以引导核酸内切酶进行基因编辑,从而产生随机突变。In the above gene editing system, the editing element is an endonuclease; the targeting element is two sgRNA sequences: sgRNA1 and sgRNA2, the partial sequence of the sgRNA2 and the sequence of the corresponding sgRNA1 are highly coincident, and the sgRNA1 is guided into the nucleic acid. The dicer is genetically edited in the DNA region where sgRNA2 is expressed, thereby randomly generating a new sgRNA, which can direct the endonuclease for gene editing, thereby generating a random mutation.
上述基因编辑系统包括CRISPR系统。The above gene editing system includes a CRISPR system.
上述基因编辑系统中,所述两种sgRNA序列分别包括一个或两个以上相同或不同的序列。In the above gene editing system, the two sgRNA sequences respectively comprise one or two identical or different sequences.
上述基因编辑系统中,所述sgRNA2的部分序列和相应sgRNA1的序列高度重合是指表达sgRNA2的基因序列可以被sgRNA1识别并且被核酸内切酶剪切。In the above gene editing system, the partial sequence of the sgRNA2 and the sequence of the corresponding sgRNA1 are highly coincident means that the gene sequence expressing sgRNA2 can be recognized by sgRNA1 and cleaved by an endonuclease.
例如,在一实施例中,某一个sgRNA2的序列包括相应sgRNA1的序列和核酸内切酶识别位点(例如,PAM序列),即sgRNA2部分序列和sgRNA1的靶点序列重合,并且在sgRNA1的靶点序列之后设置核酸内切酶识别位点,使核酸内切酶对sgRNA2基因进行剪切,然后修复后,随机生成新的sgRNA基因,并表达新的sgRNA。For example, in one embodiment, the sequence of a certain sgRNA2 comprises the sequence of the corresponding sgRNA1 and an endonuclease recognition site (eg, a PAM sequence), ie, the sgRNA2 partial sequence and the target sequence of sgRNA1 are coincident, and the target of sgRNA1 After the point sequence, the endonuclease recognition site is set, and the endonuclease cleaves the sgRNA2 gene, and then repairs, randomly generates a new sgRNA gene, and expresses a new sgRNA.
上述基因编辑系统中,所述核酸内切酶为Cas蛋白或Cpf1蛋白。In the above gene editing system, the endonuclease is a Cas protein or a Cpf1 protein.
上述基因编辑系统中,优选的,所述Cas蛋白为Cas9、Cas9n、dCas9或xCas9。In the above gene editing system, preferably, the Cas protein is Cas9, Cas9n, dCas9 or xCas9.
本发明的第二目的在于提供表达上述基因编辑系统的核酸。A second object of the present invention is to provide a nucleic acid expressing the above gene editing system.
本发明的第三目的在于提供表达上述基因编辑系统的载体。A third object of the present invention is to provide a vector expressing the above gene editing system.
所述载体为单载体或双载体;The carrier is a single carrier or a dual carrier;
所述单载体是指表达上述基因编辑系统的核酸在一个载体上,所述双载体是指表达上述基因编辑系统的核酸在两个载体上,例如,在一实施例中,核酸内切酶和两种sgRNA序列的表达基因分别位于两个载体上。The single vector refers to a nucleic acid expressing the above-described gene editing system on a vector, and the double vector refers to a nucleic acid expressing the above-described gene editing system on two vectors, for example, in one embodiment, an endonuclease and The expression genes of the two sgRNA sequences are located on two vectors, respectively.
上述载体为可以表达CRISPR系统的载体,可以为植物载体、动物载体、细菌载体或真菌载体;The above vector is a vector capable of expressing the CRISPR system, and may be a plant vector, an animal vector, a bacterial vector or a fungal vector;
上述载体为可选pHEE401E载体质粒、腺病毒载体、pCRISPR载体、pDONR207载体、lentiCRISPR载体、pCMV载体、pU6载体、T1706、pLko载体等。The above vector is an optional pHEE401E vector plasmid, an adenovirus vector, a pCRISPR vector, a pDONR207 vector, a lenti CRISPR carrier, a pCMV vector, a pU6 vector, a T1706, a pLko vector, and the like.
本发明的第四目的在于提供一种细胞中随机突变的方法,包括步骤如下:将上述核酸或上述载体导入宿主细胞中。A fourth object of the present invention is to provide a method for random mutation in a cell comprising the steps of introducing the above nucleic acid or the above vector into a host cell.
本发明的第五目的在于提供一种突变体库的构建方法,包括步骤如下:将上述核酸或上 述载体导入宿主细胞中。A fifth object of the present invention is to provide a method for constructing a mutant library comprising the steps of introducing the above nucleic acid or the above vector into a host cell.
上述方法中,所述宿主为细菌、真菌、植物或动物。In the above method, the host is a bacterium, a fungus, a plant or an animal.
上述方法中,所述宿主细胞为有性生殖生物中的生殖细胞或无性生殖生物中的可分裂细胞并成长为个体的细胞。In the above method, the host cell is a germ cell in a sexual reproductive organism or a cleavable cell in an asexual reproductive organism and grows into a cell of the individual.
上述方法中,所述方法还包括:培养宿主细胞的下一代细胞或个体;In the above method, the method further comprises: cultivating a next generation cell or an individual of the host cell;
或者进一步地,在宿主的下一代细胞或个体中按上述方法重复地导入上述核酸或上述载体。Alternatively, the nucleic acid or the above vector is repeatedly introduced into the next generation cell or individual of the host as described above.
例如,在一实施例中,在植物中,将上述核酸或上述载体导入植物卵细胞中,利用卵细胞特异启动子(或其它生殖细胞特异表达启动子)驱动Cas9,组成性表达sgRNA时,sgRNA2引导的核酸内切酶对sgRNA1基因的编辑产生的随机突变将只发生在卵细胞中,随机产生的新的sgRNA也将只在卵细胞中对植物基因组进行编辑,收获的种子即具有不同的编辑事件。一株植物能够产生成千上万粒种子,理论上,大部分种子中将含有新的sgRNA基因,如基因组有匹配序列,则将产生新的突变。For example, in one embodiment, in a plant, the nucleic acid or the vector is introduced into a plant egg cell, and Cas9 is driven by an egg cell-specific promoter (or other germ cell-specific expression promoter) to sgRNA2-directed constitutive expression of sgRNA. The random mutations generated by the endonuclease editing of the sgRNA1 gene will only occur in the egg cells, and the new sgRNA generated randomly will also edit the plant genome only in the egg cells, and the harvested seeds will have different editing events. A plant can produce tens of thousands of seeds. In theory, most seeds will contain new sgRNA genes. If the genome has matching sequences, new mutations will be produced.
在本发明中,“植物”应理解为能够进行光合作用的任何分化的多细胞生物,特别是单子叶或双子叶植物,例如:(1)粮食作物:稻属(Oryza spp.),例如稻(Oryza sativa)、阔叶稻(Oryza latifolia)、水稻(Oryza sativa)、光稃稻(Oryza glaberrima);小麦属(Triticum spp.),例如普通小麦(Triticum aestivum)、硬粒小麦(T.Turgidumssp.durum);大麦属(Hordeum spp.),例如大麦(Hordeum vulgare)、亚利桑那大麦(Hordeum arizonicum);黑麦(Secale cereale);燕麦属(Avena spp.),例如燕麦(Avena sativa)、野燕麦(Avena fatua)、比赞燕麦(Avena byzantina)、Avena fatua var.sativa、杂种燕麦(Avena hybrida);稗属(Echinochloa spp.),例如,珍珠粟(Pennisetum glaucum)、高粱(两色高粱(Sorghum bicolor)、高粱(Sorghum vulgare))、黑小麦、玉蜀黍或玉米、粟、稻(rice)、谷子、糜子、两色蜀黍(Sorghum bicolor)、黍子、荞麦属(Fagopyrum spp.)、黍(Panicum miliaceum)、小米(Setaria italica)、沼生菰(Zizania palustris)、埃塞俄比亚画眉草(Eragrostis tef)、稷(Panicum miliaceum)、龙爪稷(Eleusine coracana);(2)豆类作物:大豆属(Glycine spp.),例如大豆(Glycine max)、黄豆(Soja hispida)、Soja max)、野豌豆属(Vicia spp.)、豇豆属(Vigna spp.)、豌豆属(Pisum spp.)、芸豆(field bean)、羽扇豆属(Lupinus spp.)、蚕豆属(Vicia)、酸豆(Tamarindus indica)、兵豆(Lens culinaris)、山黧豆属(Lathyrus spp.)、扁豆属(Lablab)、蚕豆、绿豆、红豆、鹰嘴豆;(3)油料作物:花生(Arachis hypogaea)、落花生属(Arachis spp)、胡麻属(Sesamum spp.)、向日葵属(Helianthus spp.)(例如向日葵(Helianthus annuus))、油棕属(Elaeis)(例如油棕(Eiaeis guineensis)、美洲油棕(Elaeis oleifera))、大豆(soybean)、油菜 (Brassicanapus)、芸苔、芝麻、芥菜(Brassicajuncea)、油菜籽油菜(oilseedrape)、油茶、油棕、油橄榄、蓖麻、欧洲油菜(Brassica napus L.)、卡诺拉油菜(canola);(4)纤维作物:剑麻(Agave sisalana)、棉属(棉花、海岛棉(Gossypium barbadense)、陆地棉(Gossypium hirsutum))、红麻、剑麻、蕉麻、亚麻(Linum usitatissimum)、黄麻、苎麻、大麻(Cannabis sativa)、火麻;(5)水果类作物:枣属(Ziziphus spp.)、香瓜属(Cucumis spp.)、鸡蛋果(Passiflora edulis)、葡萄属(Vitis spp.)、越桔属(Vaccinium spp.)、西洋梨(Pyrus communis)、李属(Prunus spp.)、番石榴属(Psidium spp.)、石榴(Punica granatum)、苹果属(Malus spp.)、西瓜(Citrullus lanatus)、柑桔属(Citrus spp.)、无花果(Ficus carica)、金桔属(Fortunella spp.)、草莓属(Fragaria spp.)、山楂属(Crataegus spp.)、柿树属(Diospyros spp.)、红仔果(Eugenia unifora)、枇杷(Eriobotrya japonica)、龙眼(Dimocarpus longan)、番木瓜(Carica papaya)、椰子属(Cocos spp.)、阳桃(Averrhoa carambola)、狲猴桃属(Actinidia spp.)、扁桃(Prunus amygdalus)、芭蕉属(Musa spp.)(香蕉)、鳄梨属(Persea spp.)(鳄梨(Persea americana))、番石榴(Psidium guajava)、曼密苹果(Mammea americana)、芒果(Mangifera indica)、橄榄(油橄榄(Oleaeuropaea))、番木瓜(Caricapapaya)、椰子(Cocos nucifera)、凹缘金虎尾(Malpighia emarginata)、人心果(Manilkara zapota)、菠萝(Ananas comosus)、番荔枝属(Annona spp.)、柑桔树(柑桔属物种(Citrus spp.))、波罗蜜属(Artocarpus spp.)、荔枝(Litchi chinensis)、茶藨子属(Ribes spp.)、悬钩子属(Rubus spp.)、梨、桃、杏、梅、杨梅、柠檬、金橘、榴莲、橙、草莓(straw berry)、蓝莓、哈密瓜、甜瓜、椰枣、胡桃树、樱桃树;(6)根茎类作物:木薯属(Manihot spp.)、甘薯(Ipomoea batatas)、芋(Colocasia esculenta)、榨菜、洋葱、荸荠、油莎草、山药;(7)蔬菜类作物:菠菜属(Spinacia spp.)、菜豆属(Phaseolus spp.)、莴苣(Lactuca sativa)、苦瓜属(Momordica spp)、欧芹(Petroselinum crispum)、辣椒属(Capsicum spp.)、茄属(Solanum spp.)(例如马铃薯(Solanum tuberosum)、红茄(Solanum integrifolium)或蕃茄(Solanum lycopersicum))、蕃茄属(Lycopersicon spp.)(例如西红柿(Lycopersicon esculentum)、蕃茄(Lycopersicon lycopersicum)、梨形蕃茄(Lycopersicon pyriforme))、硬皮豆属(Macrotyloma spp.)、无头甘蓝(kale)、棱角丝瓜(Luffa acutangula)、小扁豆(lentil)、秋葵(okra)、洋葱(onion)、马铃薯(potato)、洋蓟(artichoke)、芦笋(asparagus)、西兰花(broccoli)、球芽甘蓝(Brussels sprouts)、卷心菜(cabbage)、胡萝卜(carrot)、花椰菜(cauliflower)、芹菜(celery)、羽衣甘蓝(collard greens)、西葫芦(squash)、冬瓜(Benincasa hispida)、石刁柏(Asparagus officinalis)、旱芹(Apium graveolens)、苋属(Amaranthus spp.)、葱属(Allium spp.)、秋葵属(Abelmoschus spp.)、苦苣(Cichorium endivia)、南瓜属(Cucurbita spp.)、芫荽(Coriandrum sativum)、埃塞 俄比亚芥(B.carinata)、萝卜(Rapbanus sativus)、芸苔属(Brassica)物种(例如例如欧洲油菜(Brassica napus)、芜菁亚种(Brassica rapa ssp.)、卡诺拉油菜(canola)、油籽油菜(oilseed rape)、芜菁油菜(turnip rape)、芥菜、甘蓝、黑芥、油菜籽油菜)、孢子甘蓝、茄科植物(茄子)、甜椒、黄瓜、丝瓜、白菜、油菜、甘蓝、葫芦、韭菜、莲、藕、生菜;(8)花卉作物:小金莲花(Tropaeolum minus)、金莲花(Tropaeolum majus)、美人蕉(Canna indica)、仙人掌属(Opuntia spp.)、万寿菊属(Tagetes spp.)、兰花、文殊兰、君子兰、朱顶红、玫瑰、月季、茉莉花、郁金香、樱花、牵牛花、金盏花、荷花、雏菊、康乃馨、矮牵牛花、郁金香、百合、梅花、水仙、迎春、报春、瑞香、山茶、白玉兰、紫玉兰、琼花、君子兰、海棠、牡丹、芍药、丁香、杜鹃、西洋杜鹃、含笑、紫荆、棣棠、锦带花、连翘、云南黄馨、金雀花、仙客来、蝴蝶兰、石斛、风信子、鸢尾、马蹄莲、金盏菊、百枝莲、四季海棠、吊钟海棠、竹节海棠、天竺葵;(9)药用作物:红花(Carthamus tinctorius)、薄荷属(Mentha spp.)、波叶大黄(Rheum rhabarbarum)、番红花(Crocus sativus)、枸杞、玉竹、黄精、知母、麦冬、川贝、郁金、砂仁、何首乌、大黄、甘草、黄芪、人参、三七、五加、当归、川芎、北柴胡、曼佗罗、洋金花、薄荷、益母草、藿香、黄芩、夏枯草、除虫菊、银杏、金鸡纳树、天然橡胶树、苜蓿、胡椒;(10)原料作物:橡胶、蓖麻(Ricinus communis)、油桐、桑、忽布、桦、桤木、漆树;(11)牧草作物:冰草属(Agropyron spp.)、车轴草属(Trifolium spp.)、芒(Miscanthus sinensis)、狼尾草属(Pennisetum sp.)、虉草(Phalaris arundinacea)、柳枝稷(Panicum virgatum)、草原草(prairiegrasses)、印度草(Indiangrass)、大须芒草(Big bluestem grass)、梯牧草(Phleum pratense)、草皮草(turf)、莎草科(高山嵩草、脚苔草(Carex pediformis)、低苔草)、苜蓿、梯牧草、紫花苜蓿、草木犀、紫云英、柽麻、田菁、红萍、水葫芦、紫穗槐、羽扇豆、三叶草、沙打旺、水浮莲、水花生、黑麦草;(12)糖料作物:甘蔗(甘蔗属物种(Saccharum spp.))、甜菜(Beta vulgaris);(13)饮料作物:大叶茶(Camellia sinensis)、茶(Camellia Sinensis)、茶树(tea)、咖啡(咖啡属物种(Coffea spp.))、可可树(Theobroma cacao)、蛇麻花(啤酒花);(14)草坪植物:固沙草(Ammophila arenaria)、早熟禾属(Poa spp.)(草地早熟禾(Poa pratensis)(蓝草))、剪股颖属物种(Agrostis spp.)(剪股颖、匍匐剪股颖(Agrostis palustris))、黑麦草属物种(Lolium spp.)(黑麦草)、羊茅属物种(Festuca spp.)(羊茅)、结缕草属物种(Zoysia spp.)(结缕草(Zoysiajaponica))、狗牙根属物种(Cynodon spp.)(百慕大草、狗牙根)、侧钝叶草(Stenotaphrum secunda tum)(圣奥古斯丁草)、雀稗属物种(Paspalum spp.)(巴哈草)、假俭草(Eremochloa ophiuroides)(百足草)、地毯草属物种(Axonopus spp.)(地毯草)、指形垂穗草(Bouteloua dactyloides)(野牛草)、垂穗草属变种物种(Bouteloua var.spp.)(格兰马草)、马 唐(Digitariasanguinalis)、香附子(Cyperusrotundus)、短叶水蜈蚣(Kyllingabrevifolia)、阿穆尔莎草(Cyperusamuricus)、加拿大飞蓬(Erigeroncanadensis)、天胡荽(Hydrocotylesibthorpioides)、鸡眼草(Kummerowiastriata)、地锦(Euphorbiahumifusa)、耕地堇菜(Violaarvensis)、白颖苔草、异穗苔草、草皮草(turf);(15)树木作物:松属(Pinus spp.)、柳属(Salix sp.)、槭树属(Acer spp.)、木槿属(Hibiscus spp.)、桉属(Eucalyptus sp.)、银杏(Ginkgo biloba)、箣竹属(Bambusa sp.)、杨属(Populus spp.)、牧豆树属(Prosopis spp.)、栎属(Quercus spp.)、刺葵属(Phoenix spp.)、山毛榉属(Fagus spp.)、吉贝(Ceiba pentandra)、樟属(Cinnamomum spp.)、黄麻属(Corchorus sp.)、南方芦苇(Phragmites australis)、酸浆属(Physalis spp.)、山蚂蝗属(Desmodium spp.)、杨、常春藤、白杨、珊瑚树、银杏、栎类、臭椿、木荷、冬青、悬铃木、女贞、大叶黄扬、落叶松、黑荆树、马尾松、思茅松,云南松、南亚松、油松、红松、黑胡桃、柠檬、悬铃木、蒲桃、珙桐、木棉、爪哇木棉、洋紫荆、羊蹄甲、雨树、合欢、龙牙花、刺桐、广玉兰、苏铁、紫薇、针叶树、乔木、灌木;(16)坚果作物:巴西栗(Bertholletia excelsea)、栗属(Castanea spp.)、榛属(Corylus spp.)、山核桃属(Carya spp.)、核桃属(Juglans spp.)、阿月浑子(Pistacia vera)、腰果(Anacardium occidentale)、澳洲坚果(全缘叶澳洲坚果(Macadamia integrifolia))、碧根果、夏威夷果、开心果、巴旦木以及产生坚果的植物;(17)其他:拟南芥、臂形草、蒺藜草、大狗尾草、牛筋草、Cadaba farinosa、藻类(algae)、Carex elata、观赏植物、大果假虎刺(Carissa macrocarpa)、菜蓟属(Cynara spp.)、野胡萝卜(Daucus carota)、薯蓣属(Dioscorea spp.)、蔗茅属(Erianthus sp.)、苇状羊茅(Festuca arundinacea)、萱草(Hemerocallis fulva)、百脉根属(Lotus spp.)、Luzula sylvatica、紫苜蓿(Medicago sativa)、草木樨属(Melilotus spp.)、黑桑(Morus nigra)、烟草属(Nicotiana spp.)、木犀榄属(Olea spp.)、鸟足豆属(Ornithopus spp.)、欧防风(Pastinaca sativa)、接骨木属(Sambucus spp.)、白芥属(Sinapis sp.)、蒲桃属(Syzygium spp.)、鸭茅状摩擦禾(Tripsacum dactyloides)、Triticosecale rimpaui、香堇(Viola odorata)等。In the present invention, "plant" is understood to mean any differentiated multicellular organism capable of photosynthesis, in particular monocotyledonous or dicotyledonous plants, for example: (1) a food crop: Oryza spp., such as rice. (Oryza sativa), Oryza latifolia, Oryza sativa, Oryza glaberrima, Triticum spp., such as Triticum aestivum, Durum wheat (T.Turgidumssp) .durum); Hordeum spp., such as Hordeum vulgare, Hordeum arizonicum; Secale cereale; Avena spp., such as oat (Avena sativa), wild oats (Avena fatua), Avena byzantina, Avena fatua var.sativa, Avena hybrida, Echinochloa spp., for example, Pennisetum glaucum, sorghum (Sorghum) Bicolor), Sorghum vulgare, black wheat, maize or corn, millet, rice, millet, hazelnut, Sorghum bicolor, scorpion, Fagopyrum spp., Panicum Mi Liaceum), Setaria italica, Zizania palustris, Eragrostis tef, Panicum miliaceum, Eleusine coracana; (2) Bean crop: Glycine Spp.), such as soybean (Glycine max), soybean (Soja hispida), Soja max), Vicia spp., Vigna spp., Pisum spp., cow bean (field bean) ), Lupinus spp., Vicia, Tamarindus indica, Lens culinaris, Lathyrus spp., Lablab, broad beans, mung beans , red beans, chickpeas; (3) oil crops: peanuts (Arachis hypogaea), Arachis spp, Sesamum spp., Helianthus spp. (such as sunflower (Helianthus annuus), Oil genus (Elaeis) (eg Eiaeis guineensis, Elaeis oleifera), soybean, rape (Brassicanapus), canola, sesame, mustard (Brassicajuncea), rapeseed (oilseedrape) , Camellia, Oil Palm, Olive Oil, Castor, Europe Brassica napus L., canola; (4) fiber crops: Agave sisalana, cotton (cotton, Gossypium barbadense, Gossypium hirsutum), red Hemp, sisal, abaca, flax (Linum usitatissimum), jute, ramie, cannabis (Cannabis sativa), hemp; (5) fruit crops: Ziziphus spp., Cucumis spp. Eggia (Passiflora edulis), Vitis spp., Vaccinium spp., Pyrus communis, Prunus spp., Psidium spp., pomegranate Punica granatum), Malus spp., Citrus autum lanatus, Citrus spp., Ficus carica, Fortunella spp., Fragaria spp. Crataegus spp., Diospyros spp., Eugenia unifora, Eriobotrya japonica, Dimocarpus longan, Carica papaya, Cocos spp. ), Averrhoa carambola, Actinidia spp., almond (Pr) Unus amygdalus), Musa spp. (banana), Persea spp. (Persea americana), guava (Psidium guajava), Manmie americana, Mangifera Indica), olive (Oleaeuropaea), papaya (Caricapapaya), coconut (Cocos nucifera), Malpighia emarginata, Manilkara zapota, Ananas comosus, Annona Spp.), citrus tree (Citrus spp.), Artocarpus spp., litchi chinensis, Ribes spp., Rubus spp. ), pear, peach, apricot, plum, bayberry, lemon, kumquat, durian, orange, strawberry (berry), blueberry, cantaloupe, melon, date palm, walnut tree, cherry tree; (6) root crop: cassava Genihot spp., Ipomoea batatas, Colocasia esculenta, mustard, onion, alfalfa, oil sedge, yam; (7) vegetable crops: spinacia spp., genus (Phaseolus) Spp.), lettuce (Lactuca sativa), Momordica spp. ), Pestelinum crispum, Capsicum spp., Solanum spp. (eg Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Lycopersicon Spp.) (eg, tomatoes (Lycopersicon esculentum), tomatoes (Lycopersicon lycopersicum, Lycopersicon pyriforme), genus Macrotyloma spp. Lentil, okra, onion, potato, artichoke, asparagus, broccoli, Brussels sprouts, cabbage ), carrot, cauliflower, celery, collard greens, squash, benincasa hispida, Asparagus officinalis, Apium graveolens, Amaranthus spp., Allium spp., Abelmoschus spp., Cichorium endivia, Cucurbita spp., Coriandrum sativum, A. B. carinata, Rapbanus sativus, Brassica species (eg, Brassica napus, Brassica rapa ssp., canola) ), oilseed rape, turnip rape, mustard, cabbage, black mustard, rapeseed rape, spore cabbage, Solanaceae (eggplant), sweet pepper, cucumber, loofah, cabbage, rape , cabbage, gourd, leeks, lotus, alfalfa, lettuce; (8) flower crops: Tropaeolum minus, Tropaeolum majus, Canna indica, Opuntia spp., marigold (Tagetes spp.), orchid, Manjusri, Clivia, arborvitae, rose, rose, jasmine, tulip, cherry blossom, morning glory, calendula, lotus, daisy, carnation, petunia, tulip, lily, plum, Narcissus, Yingchun, Primula, Ruixiang, Camellia, Magnolia, Purple Magnolia, Qionghua, Clivia, Begonia, Peony, Peony, Clove, Rhododendron, Western Rhododendron, Chixiao, Bauhinia, Poria, Jinhuahua, Forsythia, Yunnan yellow , gorse, cyclamen, phalaenopsis, sarcophagus, hyacinth, iris, calla lily, calendula, sage, sea bream, bellow, sea bream, geranium; (9) medicinal crop: red Flower (Carthamus tinctorius), Mentha spp., Rheu rhrabarbarum, Crocus sativus, Poria, Polygonatum, Polygonatum, Zhimu, Ophiopogon, Chuanbei, Yujin, sand Ren, Polygonum, Rhubarb, Licorice, Astragalus, Ginseng, Sanqi, Wujia, Angelica, Chuanxiong, Bupleurum, Mandala, Golden Flower, Peppermint, Motherwort, Musk, Astragalus, Prunella, Pyrethrum, Ginkgo, Cinchona tree, natural rubber tree, alfalfa, pepper; (10) raw material crops: rubber, ramie (Ricinus communis), tung tree, mulberry, bud, birch, eucalyptus, lacquer tree; (11) pasture crop: genus (Agropyron spp.), Trifolium spp., Miscanthus sinensis, Pennisetum sp., Phalaris arundinacea, Panicum virgatum, prairiegrasses, Indiangrass, Big blue Stem grass), Phleum pratense, turf, sedge (Carle pediformis, sedge), sedge, steppe, alfalfa, rhododendron, purple Yunying, Ramie, Tianjing, Azolla, Water Hyacinth, Amorpha, Lupin, Clover, Sandawang, Water Lotus, Water Peanut, Ryegrass; (12) Sugar Crop: Sugarcane (Saccharum species) (Saccharum spp.), Beet (Beta vulgaris); (13) Beverage crops: Camellia sinensis, Camellia Sinensis, tea, coffee (Coffea spp.), Theobroma cacao, hops (hops); (14) turf plants: Ammophila arenaria, Poa spp. (Poa pratensis (bluegrass)), cut stock Agrostis spp. (Agrostis palustris), Lolium spp. (rye), Festuca spp. (feather grass) , Zoysia spp. (Zoysia japonica), Cynodon spp. (Bermuda grass, Bermuda root) , Stenotaphrum secunda tum (St. Augustine), Paspalum spp. (Baja grass), Eremochloa ophiuroides (Hundred Grass), Carpet species (Axonopus spp .) (Carpet grass), Bouteluaa dactyloides (Buffalo grass), Phyllostachys var. sp. (Gutema grass), Digitaria sanguinalis, Aconite ( Cyperusrotundus), Kyllingabrevifolia, Cyperusamuricus, Erigeroncanadensis, Hydrocotylesibthorpioides, Kummerowistriata, Euphorbiahumifusa, Violaarvensis, White stalk, stalk, turf; (15) tree crops: Pinus spp., Salix sp., Acer spp., Hibiscus spp .), Eucalyptus sp., Ginkgo biloba, Bambusa sp., Populus spp., Prosopis spp., Quercus spp. ), Phoenix spp., Fagus spp., Kyrgyzstan (Ceiba pentandra), Cinnamomum spp., Corchorus sp., Phragmites australis, Physalis spp., Desmodium spp., Yang, Chang Ivy, poplar, coral tree, ginkgo, medlar, skunk, hibiscus, holly, sycamore, privet, big leaf huangyang, larch, black wattle, masson pine, Simao pine, Yunnan pine, South Asian pine, Chinese pine , Korean pine, black walnut, lemon, sycamore, rose apple, paulownia, kapok, Javan kapok, bauhinia, hoof, rain tree, acacia, dragon tooth, thorn, magnolia, sago, crape, conifer, tree (16) nut crops: Brazilian chestnut (Bertholletia excelsea), Castanea spp., Corylus spp., Carya spp., Juglans spp. Pistacia vera, Anacardium occidentale, macadamia (Macadamia integrifolia), pecans, macadamia, pistachios, almonds, and nuts-producing plants; (17) Other: Arabidopsis, arm grass, valerian, large Foxtail, goosegrass, Cadaba farinosa, algae, Carex elata, ornamental plants, Carissa macrocarpa, Cynara spp., Daucus carota, Dioscorea Spp.), Erianthus sp., Festuca arundinacea, Hemerocallis fulva, Lotus spp., Luzula sylvatica, Medicago sativa, hibiscus Genus (Melilotus spp.), Morus nigra, Nicotiana spp., Olea spp., Ornithopus spp., Pastinaca sativa, elderberry Genus (Sambucus spp.), Sinapis sp., Syzygium spp., Tripsacum dactyloides, Triticosecale rimpaui, Viola odorata, and the like.
本发明的有益效果Advantageous effects of the present invention
(1)本发明提供了一种新的随机突变的基因编辑系统,所述系统利用CRISPR系统可同时对多个不同位点编辑的特性,设计了两种不同sgRNA,其中一种sgRNA序列与表达另一种sgRNA的DNA区域高度重合,这导致了前者sgRNA会引导核酸内切酶在表达后者sgRNA的DNA区域进行编辑,从而随机产生了新的随机sgRNA。这些新的随机产生的sgRNA一旦与基因组DNA有匹配,就会引导核酸内切酶在这些随机位点上产生突变。利用本发明的系统,可以在细胞体内进行随机突变,是一种随机突变的新方法。(1) The present invention provides a novel random mutation gene editing system which utilizes the CRISPR system to simultaneously edit the characteristics of a plurality of different sites, and designs two different sgRNAs, one of which is sgRNA sequence and expression. The DNA region of another sgRNA is highly coincident, which results in the sgRNA of the former directing the endonuclease to edit the DNA region expressing the latter sgRNA, thereby randomly generating a new random sgRNA. These new randomly generated sgRNAs, once matched to genomic DNA, direct the endonuclease to mutate at these random sites. Using the system of the present invention, random mutations can be made in cells, a novel method of random mutation.
(2)利用本发明的基因编辑系统可以在体细胞内随机进行突变,并且可以进行不同程度的多种突变。如果利用卵细胞特异启动子驱动核酸内切酶,可以得到大量具有独立突变事件的受精卵或合子,且该过程可以在下一代重复发生,只是设计一种或几种不同的sgRNA基因编辑系统可以构建含量成千上万的随机突变体库,不用设计上千条sgRNA,此种方法效率高、成本低,可以很好的构建突变体库。(2) Mutations can be randomly performed in somatic cells using the gene editing system of the present invention, and various mutations can be performed to varying degrees. If an endogenous enzyme is driven by an egg cell-specific promoter, a large number of fertilized eggs or zygotes with independent mutation events can be obtained, and the process can be repeated in the next generation, but only one or several different sgRNA gene editing systems can be constructed. The tens of thousands of random mutant libraries do not need to design thousands of sgRNAs. This method is efficient and low-cost, and can be used to construct mutant libraries.
附图说明DRAWINGS
图1显示了sgRNA1-1和sgRNA2-1表达盒序列;Figure 1 shows the sgRNA1-1 and sgRNA2-1 expression cassette sequences;
图2显示了最终载体的表达盒结构;Figure 2 shows the structure of the expression cassette of the final vector;
图3显示了sgRNA1-1区域变化类型;Figure 3 shows the type of sgRNA1-1 region change;
图4显示了T2代出现白化苗;Figure 4 shows the emergence of albino seedlings in the T2 generation;
图5显示了4株白化苗的PDS3基因均发生了移码突变。Figure 5 shows that the PDS3 genes of four albino seedlings have undergone frameshift mutations.
具体实施方式detailed description
以下结合实例,对本发明作进一步说明。下面的说明是采用举例的方式,但是本发明的保护范围不应局限于此。The invention will be further illustrated by the following examples in connection with examples. The following description is by way of example, but the scope of protection of the present invention should not be limited thereto.
实施例1.设计构建能够自发打靶产成新的sgRNA靶点序列Example 1. Design and construction capable of spontaneous targeting to generate new sgRNA target sequences
通过直接基因合成的方法构建分别表达sgRNA1-1和sgRNA2-1的两个表达盒。sgRNA2-1将引导Cas9对表达sgRNA1-1的DNA区域进行编辑,从而产生新的sgRNA序列。其中,sgRNA1-1表达盒序列SEQ ID NO:1和sgRNA2-1表达盒序列SEQ ID NO:2如图1所示。Two expression cassettes expressing sgRNA1-1 and sgRNA2-1, respectively, were constructed by direct gene synthesis. sgRNA2-1 will direct Cas9 to edit the region of DNA that expresses sgRNA1-1, resulting in a new sgRNA sequence. Wherein, the sgRNA1-1 expression cassette sequence SEQ ID NO: 1 and the sgRNA2-1 expression cassette sequence SEQ ID NO: 2 are shown in FIG.
将两个表达盒串联在一起,克隆至pHEE401E载体{参考(1)Xing H L,Dong L,Wang Z P,et al.A CRISPR/Cas9toolkit for multiplex genome editing in plants[J].BMC Plant Biology,2014,14(1):327.(2)Wang Z P,Xing H L,Dong L,et al.Egg cell-specific promoter-controlled CRISPR/Cas9efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation[J].Genome Biology,2015,16(1):144.}中,转入农杆菌中,以供通过蘸花法转化拟南芥。其中,最终载体的表达盒结构如图2所示,载体全序列示于SEQ ID NO:3中。The two expression cassettes were ligated together and cloned into the pHEE401E vector {Reference (1) Xing H L, Dong L, Wang Z P, et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants [J]. BMC Plant Biology, 2014,14(1):327.(2)Wang Z P,Xing H L,Dong L,et al.Egg cell-specific promoter-controlled CRISPR/Cas9efficiently generate homozygous mutants for multiple target genes in Arabidopsis in a single generation[ J]. Genome Biology, 2015, 16(1): 144.}, transferred into Agrobacterium for transformation of Arabidopsis thaliana by the silk flower method. The expression cassette structure of the final vector is shown in Figure 2, and the full sequence of the vector is shown in SEQ ID NO: 3.
实施例2.对拟南芥进行转化{该部分所用方法和材料,参考(3)Chen Y,Wang Z,Ni H,Xu Y,Chen Q*,Jiang L.*2017.CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis.Science China Life Sciences 60,520-523.}Example 2. Transformation of Arabidopsis thaliana {Methods and materials used in this section, reference (3) Chen Y, Wang Z, Ni H, Xu Y, Chen Q*, Jiang L.*2017.CRISPR/Cas9-mediated base -editing system efficiently generates gain-of-function mutations in Arabidopsis.Science China Life Sciences 60,520-523.}
将构建好的载体通过冻融法转化到农杆菌GV3101菌株中之后,将菌液通过划线法涂于YEP固体培养基中(含有卡那霉素和庆大霉素),在28℃黑暗环境下培养36-48h后,挑取单菌落接种于1ml的已加入卡那霉素和庆大霉素的液体YEB培养基中,振荡培养过夜(28℃200rpm)。此时进行菌落PCR鉴定,并选取其中阳性克隆,加入到50ml锥形瓶中,加入25ml  YEP液体培养基(含有卡那霉素和庆大霉素),在28℃200rpm的条件下震荡培养,直到OD600值升高到0.8-1.0内。取菌液离心沉淀去除上清液,并用相同体积的5%的蔗糖溶液(100ml无菌去离子水加5g蔗糖)重悬菌体,并在菌液中加入SilwetL-77(0.02%)混匀。将适宜的拟南芥花序在菌液中蘸取0.5-1min后,在拟南芥苗上面遮盖黑色不透光塑料袋,创造黑暗条件,放置于22℃温室培养24h后转入光照培养,待种子成熟后收获种子。After the constructed vector was transformed into Agrobacterium GV3101 strain by freeze-thaw method, the bacterial solution was applied to YEP solid medium (containing kanamycin and gentamicin) by scribing, in a dark environment at 28 ° C. After 36-48 hours of culture, a single colony was picked and inoculated into 1 ml of liquid YEB medium to which kanamycin and gentamicin had been added, and cultured overnight with shaking (28 ° C, 200 rpm). At this time, colony PCR was performed, and the positive clones were selected, added to a 50 ml Erlenmeyer flask, and 25 ml of YEP liquid medium (containing kanamycin and gentamicin) was added, and cultured at 28 ° C and 200 rpm. Until the OD600 value rises to within 0.8-1.0. The supernatant was removed by centrifugation, and the cells were resuspended in the same volume of 5% sucrose solution (100 ml sterile deionized water plus 5 g sucrose), and Silwet L-77 (0.02%) was added to the bacterial solution to mix. . The appropriate Arabidopsis inflorescence was taken in the bacterial liquid for 0.5-1 min, and the black opaque plastic bag was covered on the Arabidopsis seedlings to create dark conditions. The cells were placed in a greenhouse at 22 ° C for 24 h and then transferred to light culture. Seeds are harvested after the seeds are ripe.
实施例3.筛选转基因拟南芥植株{该部分所用方法和材料,参考(3)Chen Y,Wang Z,Ni H,Xu Y,Chen Q*,Jiang L.*2017.CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis.Science China Life Sciences 60,520-523.}Example 3. Screening of transgenic Arabidopsis plants {Methods and materials used in this section, refer to (3) Chen Y, Wang Z, Ni H, Xu Y, Chen Q*, Jiang L.*2017.CRISPR/Cas9-mediated base -editing system efficiently generates gain-of-function mutations in Arabidopsis.Science China Life Sciences 60,520-523.}
收获的拟南芥种子经过消毒后,在含有潮霉素(25mg/L)的MS培养基上筛选转基因植株。提取T1代植株的DNA,针对sgRNA1-1和sgRNA2-1的区域进行PCR扩增并测序。选择没有编辑的T1代植株,收获T2代种子。The harvested Arabidopsis seeds were sterilized and screened for transgenic plants on MS medium containing hygromycin (25 mg/L). The DNA of the T1 plant was extracted, and the regions of sgRNA1-1 and sgRNA2-1 were subjected to PCR amplification and sequencing. T1 generation plants were selected without editing and T2 seeds were harvested.
实施例4.T2代植株出现了sgRNA序列Example 4. sgRNA sequence appeared in T2 plants
提取T2代绿苗的DNA,针对sgRNA1-1和sgRNA2-1区域进行PCR扩增并测序。结果发现sgRNA2-1区域不变,而sgRNA1-1区域由原来的CCTGACCGCCGAACCATGGC变成了多种不同的类型,从而产生了新的sgRNA,如图3所示。The DNA of the T2 generation green seedlings was extracted, and the sgRNA1-1 and sgRNA2-1 regions were subjected to PCR amplification and sequencing. As a result, it was found that the sgRNA2-1 region was unchanged, and the sgRNA1-1 region was changed from the original CCTGACCGCCGAACCATGGC to a plurality of different types, thereby producing a new sgRNA, as shown in FIG.
实施例5.T2代植株出现的新的sgRNA能够引导Cas9对拟南芥基因组进行编辑Example 5. New sgRNAs from T2 plants can guide Cas9 to edit Arabidopsis genomes
对上述收获的T2代种子在含有潮霉素的MS板上进行筛选,出现了大量的白化苗,如表1和图4所示。The above-obtained T2 seeds were screened on MS plates containing hygromycin, and a large number of albino seedlings appeared, as shown in Table 1 and Figure 4.
表1.T2代出现白化苗Table 1. Albino seedlings appear in the T2 generation
Figure PCTCN2019081184-appb-000001
Figure PCTCN2019081184-appb-000001
提取T2白化苗的DNA,针对sgRNA1-1和sgRNA2-1区域进行PCR扩增并测序。结果发现sgRNA2-1区域不变,而sgRNA1-1区域由原来的CCTGACCGCCGAACCATGGC变成了CCTGACCGCCGACCATGGC。这一新的sgRNA将引导Cas9切割拟南芥PDS3基因。对白化苗中的PDS3基因扩增测序表明,其PDS3基因在该sgRNA的靶向位点发生了移码突变,导致了PDS3基因功能的丧失,呈现出白化表型。如图5所示,对随机选取的4株白化苗的PDS3基因进行测序,结果表明均发生了移码突变。DNA of T2 albino seedlings was extracted, and PCR amplification and sequencing were performed on the sgRNA1-1 and sgRNA2-1 regions. As a result, it was found that the sgRNA2-1 region was unchanged, and the sgRNA1-1 region was changed from the original CCTGACCGCCGAACCATGGC to CCTGACCGCCGACCATGGC. This new sgRNA will direct Cas9 to cleave the Arabidopsis PDS3 gene. Sequencing of PDS3 gene amplification in albino seedlings indicated that the PDS3 gene had a frameshift mutation at the target site of the sgRNA, resulting in loss of PDS3 gene function and a whitening phenotype. As shown in Figure 5, the PDS3 genes of 4 randomly selected albino seedlings were sequenced, and the results showed that frameshift mutations occurred.
同时经过很多测试发现,当需要构建更多含量的随机突变体库时,可以设计几种不同的sgRNA基因编辑系统;或者一种sgRNA基因编辑系统的两种sgRNA序列分别包括一个或两 个以上相同或不同的序列,即sgRNA可以为三个以上,如sgRNA1包括sgRNA1-1、sgRNA1-2等等,sgRNA2包括sgRNA2-1、sgRNA2-2等等。另外,将本发明所述技术体系应用于二穗短柄草等模式植物中,或者其他前述的植物,如粮食作物、豆类作物、油料作物、纤维作物、水果类作物、根茎类作物、蔬菜类作物、花卉作物、药用作物、原料作物、牧草作物、糖料作物、饮料作物、草坪植物、树木作物、坚果作物等,也会产生相应的随机突变,在特定筛选压力(除草剂、干旱等非生物胁迫或病虫等生物胁迫)下,有望筛选出抗性突变体,且还可应用于各种动物中,因此具有良好的产业价值。At the same time, after many tests, it is found that when constructing more random mutant libraries, several different sgRNA gene editing systems can be designed; or two sgRNA sequences of an sgRNA gene editing system include one or two identical Or different sequences, that is, sgRNA may be three or more, such as sgRNA1 including sgRNA1-1, sgRNA1-2, and the like, and sgRNA2 includes sgRNA2-1, sgRNA2-2, and the like. In addition, the technical system of the present invention is applied to a model plant such as Brachypodium serrata, or other aforementioned plants, such as food crops, legume crops, oil crops, fiber crops, fruit crops, root crops, vegetables. Crops, flower crops, medicinal crops, raw crops, pasture crops, sugar crops, beverage crops, turf plants, tree crops, nut crops, etc., also produce corresponding random mutations at specific screening pressures (herbicides, drought) Under abiotic stress or biotic stress such as pests and diseases, it is expected to screen out resistant mutants, and it can also be applied to various animals, so it has good industrial value.
说明书中提及的所有出版物和专利申请均通过引用并入本文,如同每篇出版物或专利申请被单独、特别地通过引用并入本文一样。All publications and patent applications mentioned in the specification are hereby incorporated herein by reference in their entirety in their entirety in their entirety herein
尽管为清楚理解起见,前述发明已通过举例说明和实施例的方式较为详细地进行了描述,但显而易见的是,可以在所附权利要求书的范围内实施某些改变和修改,这样的改变和修改均在本发明的范围之内。Although the foregoing invention has been described in connection with the preferred embodiments and the embodiments of the embodiments Modifications are within the scope of the invention.

Claims (10)

  1. 一种随机突变的基因编辑系统,所述基因编辑系统包括靶向元件和编辑元件,所述靶向元件引导编辑元件对基因进行编辑,其特征在于,所述靶向元件可进行随机突变,从而引导编辑元件对新的靶点序列进行编辑。A randomly mutated gene editing system comprising a targeting element and an editing element, the targeting element directing an editing element to edit a gene, wherein the targeting element is capable of random mutation, thereby The editing component is guided to edit the new target sequence.
  2. 根据权利要求1的基因编辑系统,其特征在于,所述靶向元件包括蛋白质、RNA、DNA中的一种或两种的结合,或者所述靶向元件包括不同的蛋白质、不同的RNA或不同的DNA;所述编辑元件为以DNA或DNA的碱基为底物的酶。The gene editing system according to claim 1, wherein said targeting element comprises a combination of one or both of protein, RNA, DNA, or said targeting element comprises a different protein, a different RNA or a different DNA; the editing element is an enzyme that uses a base of DNA or DNA as a substrate.
  3. 根据权利要求1或2的基因编辑系统,其特征在于,所述编辑元件为核酸内切酶;所述靶向元件为两种sgRNA序列:sgRNA1和sgRNA2,所述sgRNA2的部分序列和相应sgRNA1的序列高度重合,使sgRNA1引导核酸内切酶在表达sgRNA2的DNA区域进行基因编辑,从而随机产生新的sgRNA,所述新的sgRNA可以引导核酸内切酶进行基因编辑,从而产生随机突变;优选地,所述基因编辑系统包括CRISPR系统;优选地,所述两种sgRNA序列分别包括一个或两个以上相同或不同的序列。The gene editing system according to claim 1 or 2, wherein said editing element is an endonuclease; said targeting element is two sgRNA sequences: sgRNA1 and sgRNA2, a partial sequence of said sgRNA2 and a corresponding sgRNA1 The sequences are highly coincident, allowing the sgRNA1-directed endonuclease to perform gene editing in the DNA region expressing sgRNA2, thereby randomly generating a new sgRNA, which can direct the endonuclease for gene editing, thereby generating a random mutation; preferably The gene editing system comprises a CRISPR system; preferably, the two sgRNA sequences comprise one or more identical or different sequences, respectively.
  4. 根据权利要求3的基因编辑系统,其特征在于,所述sgRNA2的部分序列和相应sgRNA1的序列高度重合是指表达sgRNA2的基因序列可以被sgRNA1识别并且被核酸内切酶剪切。The gene editing system according to claim 3, wherein the partial sequence of the sgRNA2 and the sequence of the corresponding sgRNA1 are highly coincident means that the gene sequence expressing sgRNA2 can be recognized by sgRNA1 and cleaved by an endonuclease.
  5. 根据权利要求3或4的基因编辑系统,其特征在于,所述核酸内切酶为Cas蛋白或Cpf1蛋白,其中,所述Cas蛋白,优选为Cas9、Cas9n、dCas9或xCas9。The gene editing system according to claim 3 or 4, wherein the endonuclease is a Cas protein or a Cpf1 protein, wherein the Cas protein is preferably Cas9, Cas9n, dCas9 or xCas9.
  6. 表达权利要求1-5任一项所述基因编辑系统的核酸。A nucleic acid expressing the gene editing system of any of claims 1-5.
  7. 表达权利要求1-5任一项所述基因编辑系统的载体。A vector expressing the gene editing system of any of claims 1-5.
  8. 一种细胞中随机突变的方法,其特征在于,包括步骤如下:A method of random mutation in a cell, characterized in that the steps are as follows:
    将权利要求6所述核酸或权利要求7所述的载体导入宿主细胞中,培养宿主。The nucleic acid of claim 6 or the vector of claim 7 is introduced into a host cell to culture the host.
  9. 一种突变体库的构建方法,其特征在于,包括步骤如下:A method for constructing a mutant library, characterized in that the steps are as follows:
    将权利要求6所述核酸或权利要求7所述的载体导入宿主细胞中,培养宿主。The nucleic acid of claim 6 or the vector of claim 7 is introduced into a host cell to culture the host.
  10. 根据权利要求8或9所述的方法,其特征在于,所述宿主细胞为有性生殖生物中的生殖细胞或无性生殖生物中的可分裂并能成长为个体的细胞。The method according to claim 8 or 9, wherein the host cell is a germ cell or an asexual reproductive organism in a sexual reproductive organism that is cleavable and capable of growing into an individual.
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