WO2020233670A1 - 一种在生物基因组上创制基因突变的系统及方法 - Google Patents

一种在生物基因组上创制基因突变的系统及方法 Download PDF

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WO2020233670A1
WO2020233670A1 PCT/CN2020/091567 CN2020091567W WO2020233670A1 WO 2020233670 A1 WO2020233670 A1 WO 2020233670A1 CN 2020091567 W CN2020091567 W CN 2020091567W WO 2020233670 A1 WO2020233670 A1 WO 2020233670A1
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tdt
gene
dna
mutations
cas9
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莫苏东
姜临建
李桐
陈文涛
李华荣
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青岛清原化合物有限公司
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Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a system and method for creating gene mutations on biological genomes.
  • Gene editing technology realizes precise gene editing in biological cells.
  • the technical principle is: through the combination of guide RNA (sgRNA or gRNA) and DNA endonuclease (such as Cas9, Cpf1, etc.) to form a complex of RNA and protein (nuclease) (referred to as RNP), the complex can be searched on the genome
  • the target sequence complementary to the guide RNA allows the DNA endonuclease to accurately cut the bound DNA in this region.
  • the results of shearing are varied, which can be double-stranded DNA breaks (DSB) with blunt ends or sticky ends, or single-stranded DNA breaks (Nick).
  • DSB or Nick by the cells of the organism will cause the insertion or deletion of bases (Indel), which will cause frameshift mutations with a high probability, resulting in the loss of gene function, thereby achieving precise editing of the target gene.
  • Indel insertion or deletion of bases
  • the DNA fragment may be repaired and integrated into the DSB or Nick region, thereby achieving precise DNA fragment insertion or replacement.
  • Cas9 usually causes blunt-ended DSBs, and the repair results are often manifested as Insertions or Deletes, referred to as Indels.
  • Indels Insertions or Deletes
  • Recent in-depth and systematic studies have shown that the repair results of DSB caused by Cas9 are not random (van Overbeek, M.et al.DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks.Mol.Cell 63,633-646 (2016).), large Part of the repair result is the deletion and insertion of small fragments.
  • the type of insertion is mainly based on the insertion of one base (Lemos, BRet al.CRISPR/Cas9cleavages in budding yeseast reveal) template insertions and strand-specific insertion/deletion profiles.Proc. Natl.Acad.Sci.USA 115,E2040–E2047(2018).). Because the result of the repair can be predicted by the model (Allen, Felicity, et al. "JACKS: joint analysis of CRISPR/Cas9knockout screens.” Genome research 29.3(2019): 464-471; Shou, Jia, et al.
  • Cpf1 also known as Cas12a
  • Cas12a Another type of Cpf1 (also known as Cas12a), which is widely used for gene knockout, is different from Cas9 in that Cpf1 cuts the DNA to produce DSB with sticky ends, and the repair result is mostly the deletion of longer fragments (Wang, Mugui ,Yanfei Mao,Yuming Lu,Xiaoping Tao,and Jian-kang Zhu.”Multiplex gene editing in rice using the CRISPR-Cpf1system.”Molecular plant 10,no.7(2017):1011-1013.;Xu,Rongfang,Ruiying Qin, Hao Li, Dongdong Li, Li Li, Pengcheng Wei, and Jianbo Yang. "Generation of targeted mutant rice using a CRISPR-Cpf1system.”
  • the present invention combines terminal deoxynucleotidyl transferase (Tdt) with Cas9 and the like for the first time, and Tdt adds bases without template at the end of DSB produced by Cas9 cutting.
  • Tdt terminal deoxynucleotidyl transferase
  • the present invention provides a system for creating gene mutations on biological genomes, which includes two technical components, one is an editing element that can produce breaks at specific positions in the genome, and the other is that no template can be added at the DNA break Elements of bases. Among them, the latter works with the repair mechanism in the cell and competes with each other, resulting in a diversified type of gene mutations, especially insertion and replacement gene mutations.
  • the "editing element capable of generating breaks at a specific position in the genome” is a protein-mediated targeting editing element or an RNA-mediated targeting editing element; the protein-mediated targeting editing element ZFN or TALEN is preferred, and the RNA-mediated targeting editing element is preferably CRISPR/Cas system.
  • the "element capable of adding bases without a template at the DNA break” is a terminal deoxynucleotidyl transferase Tdt; preferably, the terminal deoxynucleotidyl transferase Tdt is from various vertebrates , Such as mice, pigs, polar bears, humans and other mammals, chickens and other birds, zebrafish and other fish.
  • the terminal deoxynucleotidyl transferase Tdt has a targeting property that is enriched at DNA breaks.
  • the "Tdt has the targeting ability enriched at the DNA break" is obtained by fusing Tdt to the Cas gene, or by fusing with a targeting protein to combine with a specific sequence on the guide RNA; preferably Specifically, it is achieved by fusion with a targeting protein such as MS2 or PP7 to combine with a specific sequence on the guide RNA, such as the combined use of MS2 and sgRNA2.0.
  • the organisms described are not limited to the above-mentioned animals, but also include other eukaryotes [such as plants, fungi (such as yeast, etc.)], prokaryotes (such as bacteria, etc.) or non-cellular organisms with DNA (such as viruses, etc.) .
  • eukaryotes such as plants, fungi (such as yeast, etc.)
  • prokaryotes such as bacteria, etc.
  • non-cellular organisms with DNA such as viruses, etc.
  • the types of gene mutations include insertion type and replacement type gene mutations.
  • the insertion type and replacement type gene mutations refer to loss-of-function or gain-of-function mutations in the coding region of the gene, or in the regulatory region of the gene Generate loss of function or gain-of-function mutations.
  • the invention also provides the use of the system to create gene mutations on the biological genome.
  • the present invention further provides a method for applying the system to create gene mutations on the biological genome.
  • the gain-of-function (metasulfuron-methyl) gene mutation created by the system on the ILV2 gene of the yeast genome includes one or more mutations in K251F, M354L, L589F, F590S, F590V, and F590L , Preferably, including K251F, M354L, F590S, F590V and L589F/F590L.
  • the first letter represents the naturally occurring amino acid at a certain position in the specific sequence, the following number represents the position relative to the wild-type amino acid sequence, and the second letter represents the substitution of the natural amino acid. Became a different amino acid. As shown in Fig. 3, for example, K251F indicates that the lysine at position 251 is replaced by phenylalanine relative to the amino acid sequence of wild-type yeast. For double or multiple mutations, each mutation is separated by "/".
  • L589F/F590L indicates that relative to the amino acid sequence of wild-type yeast, leucine at position 589 is replaced by phenylalanine, and phenylalanine at position 590 is replaced by leucine. Both mutations are It also exists in the specific mutant protein.
  • plants should be understood as any differentiated multicellular organisms capable of photosynthesis, especially monocotyledonous or dicotyledonous plants, such as food crops, legumes, oil crops, and fiber crops mentioned in CN110616203A. , Fruit crops, root crops, vegetable crops, flower crops, medicinal crops, raw material crops, pasture crops, sugar crops, beverage crops, lawn plants, tree crops, nut crops, etc.
  • the excellent effect of the present invention is that the present invention combines an enzyme that can add bases without a template at the DNA break and a targeted nuclease that can produce DNA breaks, thereby achieving a variety of insertions at specific positions in the genome. And replacement gene mutations.
  • This technical route creates mutation types that cannot be generated by existing gene editing tools, provides a brand-new underlying tool for gene editing, and has huge scientific research and application value.
  • Figure 1 represents the pML104-2.0-Cas9-Tdt vector knocking out ADE2 to produce abundant insertion/replacement gene mutations.
  • AGG is PAM, -indicates the deleted base, and the inserted base is bolded.
  • Figure 2 represents the positions of the PAM regions corresponding to the four targets in SEQ ID NO. 2, which are shown in bold and underlined.
  • Figure 3 represents that the pML104-2.0-Cas9-Tdt vector produced alternative anti-metsulfuron mutations at the four targets.
  • WT is wild-type
  • the mutated bases are indicated in italics
  • the first line of bases under the target X/Y/Z/P represents the part of SEQ ID NO.2 sequence that contains the corresponding target
  • the remaining lines represent the base The corresponding sequence after base mutation.
  • Figure 4 represents the sequence alignment of Tdt genes from different species. Among them, mouse Tdt (UniProtKB serial number: P09838), mouse Tdt-L (UniProtKB serial number: P09838-1), polar bear Tdt (UniProtKB serial number: A0A384CWZ3), pig Tdt (UniProtKB serial number: F1SBG2), human Tdt (UniProtKB serial number: P04053), chicken Tdt (UniProtKB serial number: F1P317), zebrafish Tdt (UniProtKB serial number: Q5J2Q9).
  • mouse Tdt UniProtKB serial number: P09838
  • mouse Tdt-L UniProtKB serial number: P09838-1
  • polar bear Tdt UniProtKB serial number: A0A384CWZ3
  • pig Tdt UniProtKB serial number: F1SBG2
  • human Tdt UniPro
  • Figure 5 represents the results of targeting and repairing Cas9 and Cas9+Tdt at the same site on the rice PDS gene. The results show that Cas9+Tdt has more insertion type mutations.
  • Figure 6 represents the results of targeting and repairing Cas9 and Cas9+Tdt at the same site on rice HPPD gene. The results show that Cas9+Tdt has more insertion type mutations.
  • T2A-MS2-linker-NLS-linker-Tdt Use the amino acid sequence corresponding to T2A-MS2-linker-NLS-linker-Tdt in the sequence>Cas9-NLS-T2A-MS2-linker-NLS-linker-Tdt (the amino acid sequence is shown in SEQ ID NO.1) with rice After codon optimization, it was synthesized by GenScript, where Tdt refers to mouse Tdt (UniProtKB sequence number: P09838).
  • the pML104 vector (Addgene Plasmid#67638; http://n2t.net/addgene:67638; RRID: Addgene_67638) is used as the backbone for transformation.
  • the Cas9 gene expression cassette in this vector has the promoter pTDH3, the terminator is tADH1, and sgRNA expression
  • the box promoter is pSNR52 and the terminator tSUP4.
  • the synthesized T2A-MS2-linker-NLS-linker-Tdt gene element was cloned into the 3'end of Cas9 gene by in-fusion method to form a complete Cas9-NLS- T2A-MS2-linker-NLS-linker-Tdt gene (its amino acid sequence is shown in SEQ ID NO. 1) expression cassette, and the vector is named pML104-2.0-Cas9-Tdt.
  • the NLS-linker-Tdt gene was directly fused to the 3'end of the Cas9 gene and named pML104-2.0-Cas9-Tdt-fusion.
  • the Cas9 gene was replaced with the MS2-linker-NLS-linker-Tdt gene and named pML104-2.0-Tdt.
  • the target sequence on the yeast ADE2 gene was selected and cloned into the vector pML104-2.0-Cas9-Tdt, pML104-2.0-Cas9-Tdt-fusion, pML104-2.0-Tdt, pML104-2.0-Cas9.
  • pML104-2.0-Tdt and pML104-2.0-Cas9 are the control group
  • pML104-2.0-Cas9-Tdt and pML104-2.0-Cas9-Tdt-fusion are the experimental groups.
  • the diploid yeast strain INVSC1 (MATa his3 ⁇ 1 leu2 trp1-289 ura3-52/MAT ⁇ his3 ⁇ 1 leu2 trp1-289 ura3-52; purchased by Shanghai Weidi Biotechnology Company) was used as the transformation material to prepare yeast competence.
  • Three plasmids containing ADE2 target sequence (pML104-2.0-Cas9-Tdt, pML104-2.0-Tdt, pML104-2.0-Cas9) were transformed into yeast competent cells by chemically mediated methods.
  • yeast competence and transformation methods see Laughery, MF, Hunter, T., Brown, A., Hoopes, J., Ostbye, T., & Shumaker, T., et al. (2015).
  • New vectors for simple and streamlined crispr-cas9 genome editing in Saccharomyces cerevisiae.Yeast,32(12),711-720. The transformed competent cells were plated on uracil-deficient YNB medium (containing 0.01 mg/L adenine) for selection. The selected single clones were transferred to uracil-deficient YNB medium (without adenine) to grow. Among them, pML104-2.0-Tdt vector did not appear red clones, while pML104-2.0-Cas9-Tdt, pML104-2.0-Cas9, pML104-2.0-Cas9-Tdt-fusion vector appeared red clones. The red cloned DNA was extracted, the ADE2 gene fragment containing the target region was amplified, and the product was sequenced for one generation.
  • the first-generation sequencing results in Figure 1 show that the pML104-2.0-Cas9, pML104-2.0-Cas9-Tdt, and pML104-2.0-Cas9-Tdt-fusion vectors have successfully knocked out the ADE2 gene.
  • the genotypes produced by pML104-2.0-Cas9 knocking out ADE2 are very monotonous, with only two types, +1 and -1.
  • the pML104-2.0-Cas9-Tdt vector knocked out ADE2 to produce very rich genotypes, most of which are repair types where deletion and insertion occur simultaneously.
  • two clones showed a genotype of +3/-3, resulting in non-frameshift substitution gene mutations.
  • the four target sequences (respectively target X) on the ILV2 gene (its DNA and amino acid sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3; NCBI accession number: NC_001145) , Y, Z, P; the PAM regions corresponding to each target are marked in bold underlined in SEQ ID NO. 2, as shown in Figure 2) cloned into pML104-2.0-Cas9-Tdt, pML104-2.0-Tdt , PML104-2.0-Cas9.
  • Three vectors corresponding to each target were transformed into competent cells prepared with diploid yeast strain INVSC1 and haploid yeast BY4741.
  • the transformed competent state was spread on a medium containing 3 mg/L metsulfuron and cultured at 28°C. After 6 days, the yeast transformed with pML104-2.0-Cas9-Tdt-fusion vector showed a large number of resistant clones on the plate containing metsulfuron. Extract the yeast single clone DNA and sequence the corresponding amplicons. The results showed that the pML104-2.0-Cas9-Tdt vector produced a large number of substitutional anti-metsulfuron mutations at the four target positions (as shown in Figure 3). It should be pointed out that, in addition to including the reported resistance mutations G116S, A117V, K251T, M354K, M354V, F590C, etc.
  • Example 5 Tdt can change NHEJ repair results in rice cells, and a higher percentage of insertion type repair results appear.
  • Agarose gel electrophoresis is used to detect and cut the gel to recover fragments.
  • NEB Sac1-HF endonuclease was used to digest the pHUE411 vector under the following conditions:
  • each bottle can contain 20 seeds.
  • Protoplasts can be prepared by incubating at 26°C and 12h light for about 10 days.
  • the concentration of the protoplasts is about 2 ⁇ 10 6 /mL.
  • the CTAB method was used with some modifications. The specific method is as follows: Centrifuge the protoplasts and discard the supernatant, add 500 ⁇ L DNA extract, shake and mix, and incubate in a 65°C water bath for 1 hour; add the samples after the water bath is cooled, etc. Volume of chloroform, mix upside down and centrifuge at 10,000rpm for 10 minutes; take 400 ⁇ L of supernatant and transfer to a new 1.5mL centrifuge tube, add 1mL 70% (v/v) ethanol into -20°C precipitation for 20 minutes; Centrifuge at 12,000 rpm for 15 minutes to precipitate DNA. After the precipitate is air-dried, add 50 ⁇ L of ultrapure water to dissolve it and store at -20°C for later use.
  • the length is between 300-1000 bp.

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Abstract

提供了一种在生物基因组上创制基因突变的系统及方法，该系统包括两个技术组分，一是能够在基因组的特定位置上产生断裂的编辑元件，另一个是能够在DNA断口处无模板添加碱基的元件。通过将能够在DNA断口处无模板地添加碱基的酶与能够产生DNA断裂的靶向核酸酶组合在一起，实现了在基因组特定位置上产生多样化的插入和替换型的基因突变。

Description

一种在生物基因组上创制基因突变的系统及方法 技术领域
本发明属于生物技术领域，具体涉及一种在生物基因组上创制基因突变的系统及方法。
背景技术
基因编辑技术，特别是CRISPR/Cas技术，在生物细胞中实现了精准的基因编辑。其技术原理是：通过引导RNA(sgRNA或gRNA)与DNA内切酶(如Cas9、Cpf1等)结合形成RNA和蛋白(核酸酶)的复合物(简称RNP)，该复合物可以在基因组上搜索与引导RNA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)，大概率地造成移码突变，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。
不同的Cas内切酶会造成不同类型的DSB，生物体中在无模板条件下的修复结果也不相同。例如，Cas9通常会造成平末端的DSB，而对其的修复结果常常表现为插入(Insertions)或删除(Deletions)，简称Indels。近期深入系统的研究表明针对Cas9造成的DSB的修复结果并非随机(van Overbeek,M.et al.DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks.Mol.Cell 63,633–646(2016).)，大部分的修复结果是小片段的删除和插入，其中插入的类型以插入一个碱基为主(Lemos,B.R.et al.CRISPR/Cas9cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.Proc.Natl.Acad.Sci.USA 115,E2040–E2047(2018).)。由于修复的结果可以通过模型来预测(Allen,Felicity,et al."JACKS:joint analysis of CRISPR/Cas9knockout screens."Genome research 29.3(2019):464-471；Shou,Jia,et al."Precise and predictable CRISPR chromosomal rearrangements reveal principles of Cas9-mediated nucleotide insertion."Molecular cell 71.4(2018):498-509；Shen,Max W.,et al."Predictable and precise template-free CRISPR editing of pathogenic variants."Nature 563.7733(2018):646.；Chakrabarti,Anob M.,et al."Target-specific precision of CRISPR-mediated genome editing."Molecular cell 73.4(2019):699-713.Iyer S,Suresh S,Guo D,et al.Precise therapeutic gene correction by a simple nuclease-induced double-stranded break[J].Nature,2019:1.)在特定的位点上通过无模板的Cas9修复大概率地实现了精准的修复。另外一种广泛使用用于基因敲除的Cpf1(又称Cas12a)，和Cas9不同的是，Cpf1在DNA上切割产 生了粘性末端的DSB，其修复结果大多是更长片段的删除(Wang,Mugui,Yanfei Mao,Yuming Lu,Xiaoping Tao,and Jian-kang Zhu."Multiplex gene editing in rice using the CRISPR-Cpf1system."Molecular plant 10,no.7(2017):1011-1013.；Xu,Rongfang,Ruiying Qin,Hao Li,Dongdong Li,Li Li,Pengcheng Wei,and Jianbo Yang."Generation of targeted mutant rice using a CRISPR‐Cpf1system."Plant biotechnology journal 15,no.6(2017):713-717.)。
鉴于Cas9及Cpf1介导的无模板修复基本无法产生碱基替换式的修复，单碱基编辑的技术体系为此提供了一条有效的解决方案。通过将Cas9n(D10A)上融合胞嘧啶脱氨酶，几个不同的课题组先后独立实现了C到T的单碱基替换(Nishida K,Arazoe T,Yachie N,et al.Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems[J].Science,2016,353(6305):aaf8729.；Komor,A.C.,Kim,Y.B.,Packer,M.S.,Zuris,J.A.,&Liu,D.R.(2016).Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature,533(7603),420；Hess,G.T.,Frésard,L.,Han,K.,Lee,C.H.,Li,A.,Cimprich,K.A.,...&Bassik,M.C.(2016).Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.Nature methods,13(12),1036.；Ma Y,Zhang J,Yin W,et al.Targeted AID-mediated mutagenesis(TAM)enables efficient genomic diversification in mammalian cells[J].Nature methods,2016,13(12):1029.)。文献(Gaudelli,N.M.,Komor,A.C.,Rees,H.A.,Packer,M.S.,Badran,A.H.,Bryson,D.I.,&Liu,D.R.(2017).Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage.Nature,551(7681),464.)将腺嘌呤脱氨酶和Cas9n(D10A)融合，实现了精准的A到G的单碱基编辑。但是这两种单碱基编辑技术的局限是只能实现A到G或C到T的突变，很少产生A到T/C或C到G/A等情形的突变。
发明内容
本发明首次将末端脱氧核苷酸转移酶(terminal deoxynucleotidyl transferase,Tdt)与Cas9等结合在一起，Tdt在Cas9切割产生的DSB末端无模板地添加碱基。鉴于Cas9等敲除修复结果大多是碱基的删除，Tdt的作用正好与之相反，从而形成了大概率的替换和插入类型的修复事件。
本发明采用的技术方案如下：
本发明提供了一种在生物基因组上创制基因突变的系统，其包括两个技术组分，一是能够在基因组的特定位置上产生断裂的编辑元件，另一个是能够在DNA断口处无模板添加碱基的元件。其中，后者与细胞内的修复机制共同作用，相互竞争，从而产生了多元化的基因突变类型，尤其是插入型和替换型基因突变。
上述系统中，所述的“能够在基因组的特定位置上产生断裂的编辑元件”是蛋白介导靶向 的编辑元件或RNA介导靶向的编辑元件；所述蛋白介导靶向的编辑元件优选ZFN或TALEN等，所述RNA介导靶向的编辑元件优选CRISPR/Cas系统等。
上述系统中，所述的“能够在DNA断口处无模板添加碱基的元件”是末端脱氧核苷酸转移酶Tdt；优选地，所述末端脱氧核苷酸转移酶Tdt来自于各种脊椎动物，如鼠、猪、北极熊、人等哺乳动物，鸡等鸟类，斑马鱼等鱼类。
上述系统中，所述末端脱氧核苷酸转移酶Tdt具有在DNA断口处富集的靶向性。
上述系统中，所述的“Tdt具有在DNA断口处富集的靶向性”是通过将Tdt融合到Cas基因上获得，或通过与靶向蛋白融合来结合引导RNA上的特定序列实现；优选地，通过与MS2或PP7等靶向蛋白融合来结合引导RNA上的特定序列实现，例如MS2和sgRNA2.0的配合使用。
上述系统中，所述的生物不仅限于上述动物，还包括其他真核生物[如植物、真菌(如酵母等)]，原核生物(如细菌等)或具有DNA的非细胞生物(如病毒等)。
上述系统中，所述的基因突变类型包括插入型和替换型基因突变。
上述系统中，所述的插入型和替换型基因突变是指在基因的编码区产生功能缺失(loss-of-function)或功能获得型(gain-of-function)突变，或者在基因的调控区产生功能缺失或功能获得型突变。
本发明还提供了所述系统在生物基因组上创制基因突变的用途。
本发明进一步还提供了一种应用所述系统在生物基因组上创制基因突变的方法。
在一个实施方案中，所述系统在酵母基因组的ILV2基因上创制了的功能获得型(抗甲嘧磺隆)基因突变包括K251F、M354L、L589F、F590S、F590V和F590L中的一个或多个突变，优选地，包括K251F、M354L、F590S、F590V和L589F/F590L。
对于说明书中所用的有关氨基酸取代的术语，第一个字母代表特定序列某一位置上天然存在的氨基酸，后面的数字代表相对于野生型氨基酸序列的位置，第二个字母代表取代该天然氨基酸变成了不同的氨基酸。如图3所示，譬如K251F表示相对于野生型酵母的氨基酸序列而言，第251位的赖氨酸被苯丙氨酸取代。对于双重或多重突变，各突变之间以“/”隔开。例如，L589F/F590L表示相对于野生型酵母的氨基酸序列而言，第589位的亮氨酸被苯丙氨酸取代，第590位的苯丙氨酸被亮氨酸取代，全部两个突变均同时存在于所述具体的突变型蛋白内。
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CN110616203A中提到的粮食作物、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。
本发明的优异效果在于：本发明将能够在DNA断口处无模板地添加碱基的酶与能够产生DNA断裂的靶向核酸酶组合在一起，从而实现了在基因组特定位置上产生多样化的插入和替换型的基因突变。这一技术路线创制了现有基因编辑工具无法产生的突变类型，为基因编辑提供了全新的底层工具，具有巨大的科研和应用价值。
附图说明
图1代表pML104-2.0-Cas9-Tdt载体敲除ADE2产生丰富的插入/替换型基因突变。其中， AGG为PAM，-表示删除的碱基，插入的碱基加粗表示。
图2代表四个靶点相应的PAM区在SEQ ID NO.2中的位置，以加粗及下划线表示。
图3代表pML104-2.0-Cas9-Tdt载体在四个靶点产生了替换型的抗甲嘧磺隆突变。其中，WT为野生型，突变的碱基以斜体表示，靶点X/Y/Z/P下列的第一行碱基分别代表包含相应靶点的部分SEQ ID NO.2序列，其余各行代表碱基发生突变后的对应序列。
图4代表不同物种Tdt基因的序列比对。其中，小鼠Tdt(UniProtKB序列号：P09838)，小鼠Tdt-L(UniProtKB序列号：P09838-1)，北极熊Tdt(UniProtKB序列号：A0A384CWZ3)，猪Tdt(UniProtKB序列号：F1SBG2)，人类Tdt(UniProtKB序列号：P04053)，鸡Tdt(UniProtKB序列号：F1P317)，斑马鱼Tdt(UniProtKB序列号：Q5J2Q9)。
图5代表Cas9及Cas9+Tdt在水稻PDS基因上同一位点打靶修复结果。结果可以看出Cas9+Tdt出现了更多插入类型的突变。
图6代表Cas9及Cas9+Tdt在水稻HPPD基因上同一位点打靶修复结果。结果可以看出Cas9+Tdt出现了更多插入类型的突变。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。
实施例1 Tdt-Cas9融合载体的构建
将序列>Cas9-NLS-T2A-MS2-linker-NLS-linker-Tdt(其氨基酸序列如SEQ ID NO.1所示)中T2A-MS2-linker-NLS-linker-Tdt所对应的氨基酸序列用水稻密码子优化后，由金斯瑞公司合成，其中，Tdt是指小鼠Tdt(UniProtKB序列号：P09838)。以pML104载体(Addgene plasmid#67638；http://n2t.net/addgene:67638；RRID:Addgene_67638)为骨架进行改造，该载体中Cas9基因表达框的启动子为pTDH3，终止子为tADH1，sgRNA表达框启动子为pSNR52，终止子tSUP4。首先将其中的sgRNA元件替换成sgRNA2.0元件，命名为pML104-2.0-Cas9。将 pML104-2.0-Cas9载体用BamHI酶切后，把合成的T2A-MS2-linker-NLS-linker-Tdt基因元件通过in-fusion方法克隆到Cas9基因的3’端，形成完整的Cas9-NLS-T2A-MS2-linker-NLS-linker-Tdt基因(其氨基酸序列如SEQ ID NO.1所示)表达框，该载体命名为pML104-2.0-Cas9-Tdt。以pML104-2.0为骨架，将NLS-linker-Tdt基因直接融合到Cas9基因3’端，命名为pML104-2.0-Cas9-Tdt-fusion。以pML104-2.0为骨架，将其中Cas9基因替换成MS2-linker-NLS-linker-Tdt基因，命名为pML104-2.0-Tdt。
选取酵母ADE2基因上的靶点序列，将其分别克隆到载体中pML104-2.0-Cas9-Tdt，pML104-2.0-Cas9-Tdt-fusion，pML104-2.0-Tdt，pML104-2.0-Cas9中。其中pML104-2.0-Tdt，pML104-2.0-Cas9为对照组，pML104-2.0-Cas9-Tdt和pML104-2.0-Cas9-Tdt-fusion为实验组。
实施例2酵母的转化
以二倍体酵母菌株INVSC1(MATa his3Δ1 leu2 trp1-289 ura3-52/MATα his3Δ1 leu2 trp1-289 ura3-52；上海维地生物科技公司购买)为转化材料，制备酵母感受态。将含有ADE2靶点序列的三种质粒(pML104-2.0-Cas9-Tdt，pML104-2.0-Tdt，pML104-2.0-Cas9)，通过化学介导的方法转化酵母感受态细胞。制备酵母感受态和转化方法参见Laughery,M.F.,Hunter,T.,Brown,A.,Hoopes,J.,Ostbye,T.,&Shumaker,T.,et al.(2015).New vectors for simple and streamlined crispr-cas9 genome editing in Saccharomyces cerevisiae.Yeast,32(12),711-720.。转化后的感受态铺在尿嘧啶缺失YNB培养基(含有0.01mg/L腺嘌呤)上筛选。筛选到的单克隆，转到尿嘧啶缺失YNB培养基(不含腺嘌呤)上生长。其中pML104-2.0-Tdt载体，未出现红色克隆，而pML104-2.0-Cas9-Tdt，pML104-2.0-Cas9，pML104-2.0-Cas9-Tdt-fusion载体出现了红色克隆。提取红色克隆的DNA，扩增含有靶点区域的ADE2基因片段，产物进行一代测序。
实施例3酵母突变体基因型的鉴定
图1中一代测序结果表明，pML104-2.0-Cas9，pML104-2.0-Cas9-Tdt，pML104-2.0-Cas9-Tdt-fusion载体均成功敲除了ADE2基因。但是，pML104-2.0-Cas9载体敲除ADE2后产生的基因型非常单调，只有+1和-1两种类型。而pML104-2.0-Cas9-Tdt载体敲除ADE2后产生的基因型非常丰富，多数是删除和插入同时发生的修复类型。特别是，有两个克隆出现了+3/-3的基因型，导致了非移码替换型的基因突变。虽然pML104-2.0-Cas9-Tdt-fusion产生的红色克隆数目最少，但是测序结果表明产生了替换型突变。这些结果表明，Tdt的存在，尤其是以MS2系统赋予Tdt靶向性，高效率地改变了Cas9介导的NHEJ修复结果，导致了大概率的插入和替换型基因突变的产生。
实施例4在酵母中创制新的抗甲嘧磺隆的ILV2突变基因
按照实施例1的方法将ILV2基因(其DNA及氨基酸序列分别如SEQ ID NO.2和SEQ ID NO.3所示；NCBI accession number:NC_001145)上的四个靶点序列(分别为靶点X，Y，Z，P；各个靶点相应的PAM区在SEQ ID NO.2中以粗体下划线的形式标注，如图2所示)克隆到pML104-2.0-Cas9-Tdt，pML104-2.0-Tdt，pML104-2.0-Cas9中。将每个靶点对应的三个载体分别转化以二倍体酵母菌株INVSC1及单倍体酵母BY4741制备的感受态细胞。转化后的感受态，涂布在含有3mg/L甲嘧磺隆的培养基上，28℃培养。6天后，pML104-2.0-Cas9-Tdt-fusion载体转化的酵母在含有甲嘧磺隆的平板上出现了大量抗性克隆。提取酵母单克隆的DNA，对相应的扩增子进行测序。结果表明pML104-2.0-Cas9-Tdt载体在四个靶点位置均产生了大量替换型抗甲嘧磺隆的突变(如图3所示)。需要特别指出的是，除包括已经报道过的抗性突变G116S，A117V，K251T，M354K，M354V，F590C等(MAZUR,BARBARA J.,AND S.CARL FALCO."THE DEVELOPMENT OF HERBICIDE RESISTANT CROPS."Annual Review of Plant Biology40.1(1989):441-470.)，还包括新突变K251F，M354L，F590S，F590V，L589F/F590L等。这一结果表明Tdt与Cas9相结合是创制大量替换型突变的高效工具。
实施例5 Tdt在水稻细胞中能够改变NHEJ修复结果，出现了更高比例的插入类型修复结果。
1、设计并构建打靶载体
(1)选择OsPDS、OsHPPD等合适靶点，以下为靶点序列：
Figure PCTCN2020091567-appb-000001
(2)根据靶点序列和载体pHUE411及pHUE411-TDT载体序列，设计了以下载体构建及检测所需引物：
Figure PCTCN2020091567-appb-000002
Figure PCTCN2020091567-appb-000003
(3)构建pHUE411-TDT载体
首先，使用上述载体构建引物扩增TDT片段，反应条件为：
Figure PCTCN2020091567-appb-000004
建立PCR反应，一般反应条件是：
Figure PCTCN2020091567-appb-000005
琼脂糖凝胶电泳检测，并切胶回收片段。
随后，使用NEB Sac1-HF内切酶酶切pHUE411载体，条件如下：
Figure PCTCN2020091567-appb-000006
对上述产物切胶回收。
接下来IN-fusion无缝克隆片段与载体：
Figure PCTCN2020091567-appb-000007
(4)上述反应后产物进行转化。转化方法为：
将-80℃保存的E.coli感受态细胞置于冰中融化；待感受态细胞刚解冻时，加入10μL的连接产物，轻弹混匀后放于冰上，冰浴30min。42℃热激90s，立即置于冰上，放置2min。加入800μL的LB液体培养基，200rpm，37℃振荡培养1h后，8,000rpm离心1min收集菌体。去上清，留100μL重悬菌体，将全部菌液涂布在含相应抗生素的LB平板上，37℃倒置培养过夜，菌落PCR并测序鉴定阳性克隆。
(5)pHUE411-TDT载体构建完成后，与pHUE411载体一起插入上述靶点。
靶点退火形成双链后，建立边切边连反应，条件如下：
Figure PCTCN2020091567-appb-000008
对上述产物进行如下反应：2min 37℃，5min 16℃，共计20个循环。
(6)上述反应后产物进行转化。转化方法为：
将-80℃保存的E.coli感受态细胞置于冰中融化；待感受态细胞刚解冻时，加入10μL的连接产物，轻弹混匀后放于冰上，冰浴30min。42℃热激90s，立即置于冰上，放置2min。加入800μL的LB液体培养基，200rpm，37℃振荡培养1h后，8,000rpm离心1min收集菌体。去上清，留100μL重悬菌体，将全部菌液涂布在含相应抗生素的LB平板上，37℃倒置培养过夜，菌落PCR并测序鉴定阳性克隆。
载体标注：
Figure PCTCN2020091567-appb-000009
2、制备高纯度、高浓度质粒
本实验，使用的是Promega的质粒提取试剂盒(Midipreps DNA Purification System,Promega,A7640)。具体步骤为：
(1)向含有相应抗生素的300mL液体LB培养基中加5mL大肠杆菌，200rpm，37℃摇 菌12-16h。
(2)将上述菌液置于500mL离心管中，5,000g离心菌液10min，弃上清。
(3)加3mL CRS重悬细胞沉淀，涡旋混匀。
(4)加3mL CLS，缓慢颠倒混匀，时间不宜超过5min。
(5)加3mL中和液(Neutralization Solution)，颠倒混匀，至颜色变清澈透明。
(6)14,000g离心15min，如未形成紧实沉淀可再次离心15min。
(7)转移上清液至新的50mL离心管中，避免吸入白色沉淀。
(8)加10mL DNA净化树脂(Purification Resin，用前剧烈摇匀)，混匀。
(9)将Resin/DNA混合液倒入过滤柱中，真空泵负压法(0.05MPa)。
(10)加15mL洗柱液(Column Wash Solution,CWS)到过滤柱中，抽真空。
(11)加15mL CWS，重复抽真空一次；溶液全部通过滤柱后延长抽30s。
(12)切断过滤柱，将其转移至1.5mL离心管中，12,000g离心2min，移除残留液体，将过滤柱转移至新的1.5mL离心管中。
(13)加200μL预热至70℃灭菌水，静置2min。
(14)12,000g，离心2min，洗脱质粒DNA；浓度一般在1μg/μL左右。
3、原生质体的制备
首先准备原生质体用的水稻幼苗，品种为日本晴(Nipponbare)。水稻种子首先去壳，去壳的种子用75％乙醇漂洗1min，用5％(v/v)的次氯酸钠处理20min，然后用无菌水洗涤5次以上，放在超净台中吹干后放在盛有1/2MS的培养基的组培瓶中，每瓶可放20粒种子。26℃，12h光照培养10d左右，就可以进行原生质体的制备。
水稻原生质体制备以及PEG介导的转化在已经发表的方法(Bart et al.,2006)基础上进行了部分修改，所使用的试剂见下表。具体的步骤如下：
(1)选取幼苗叶鞘部分，用锋利的吉利剃须刀片切成约1mm的碎片，放在0.6M甘露醇和MES培养液中备用。将全部材料切好后转入20mL酶解液中，用锡箔纸包好置于28℃摇床(脱色摇床，速度10rpm)中，50rpm避光酶解约4个小时，最后2分钟将转速提高至100rpm；
(2)酶解结束后，加入等体积的W5，水平摇动10s，释放原生质体。酶解后的细胞经300目的筛子过滤后，150g离心5分钟收集原生质体；
(3)利用W5溶液漂洗细胞两次，150g离心5分钟收集原生质体；
(4)用适量的MMG溶液重悬原生质体，原生质体浓度约为2×10 6/mL。
4、原生质体的转化
(1)取上述200μL MMG重悬的原生质体加入无内毒素的高质量质粒DNA(10-20μg)，轻弹混匀。所转质粒如下：
Figure PCTCN2020091567-appb-000010
(2)加入等体积的40％(w/v)PEG溶液，轻弹混匀，28℃避光静置15分钟；
(3)诱导转化结束后缓慢加1.5mL W5溶液，轻弹混匀细胞，150g离心3分钟收集细胞，重复此步骤一次；
(4)加入1.5mL W5溶液重悬细胞，置于28℃培养箱中避光培养12-16小时，若用于提取原生质体基因组DNA，需培养48-60h。
5、基因组打靶情况检测
(1)首先需要提取原生质体DNA。使用CTAB法并进行部分修改，具体的方法如下：原生质体离心后弃上清，加入500μL DNA提取液，振荡混匀，置于65℃水浴锅中孵育1小时；水浴后的样品冷却后加入等体积的氯仿，颠倒混匀后10,000rpm离心10分钟；取400μL上清转移到一个新的1.5mL离心管中，加入1mL 70％(v/v)的乙醇放入-20℃沉淀20分钟；用12,000rpm离心15分钟沉淀DNA，待沉淀晾干后加入50μL超纯水溶解，保存于-20℃备用。
(2)利用上述检测引物，扩增含有靶位点的片段，长度为300-1000bp之间。
Figure PCTCN2020091567-appb-000011
(3)建立PCR反应，一般反应条件是：
Figure PCTCN2020091567-appb-000012
Figure PCTCN2020091567-appb-000013
(4)琼脂糖凝胶电泳检测并送测序进一步验证。
6、检测结果汇总
对获得Sanger测序结果进行分析，得到如图5-6所示结果。
如图5所示，在水稻PDS位点，可以看出，Cas9切割后的修复类型在三个样品中，均没有出现+2bp及以上的结果，主要以+1bp和短片段的删除为主。而在Cas9+Tdt处理下，虽然+1bp也是主要的修复结果，但是却出现了+2，+3甚至+4bp的修复类型。这表明Tdt在水稻细胞中Cas9引起的DNA断口处能够随机添加碱基。在水稻HPPD位点同样如此，如图6所示。
同时经过很多测试发现，采用其他不同来源的末端脱氧核苷酸转移酶Tdt(如图4所示)与Cas9相结合同样能创制大量的替换型突变，因此具有良好的产业价值。
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。
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。

Claims (10)

  1. 一种在生物基因组上创制基因突变的系统，其特征在于，包括两个技术组分，一是能够在基因组的特定位置上产生断裂的编辑元件，另一个是能够在DNA断口处无模板添加碱基的元件。
  2. 根据权利要求1所述的系统，其特征在于，所述的“能够在基因组的特定位置上产生断裂的编辑元件”是蛋白介导靶向的编辑元件或RNA介导靶向的编辑元件；所述蛋白介导靶向的编辑元件优选ZFN或TALEN，所述RNA介导靶向的编辑元件优选CRISPR/Cas系统。
  3. 根据权利要求1或2所述的系统，其特征在于，所述的“能够在DNA断口处无模板添加碱基的元件”是末端脱氧核苷酸转移酶Tdt；优选地，所述末端脱氧核苷酸转移酶Tdt来自于脊椎动物。
  4. 根据权利要求3所述的系统，其特征在于，所述末端脱氧核苷酸转移酶Tdt具有在DNA断口处富集的靶向性。
  5. 根据权利要求4所述的系统，其特征在于，所述的“Tdt具有在DNA断口处富集的靶向性”是通过将Tdt融合到Cas基因上获得，或通过与靶向蛋白融合来结合引导RNA上的特定序列实现；优选地，通过与MS2或PP7靶向蛋白融合来结合引导RNA上的特定序列实现，例如MS2和sgRNA2.0的配合使用。
  6. 根据权利要求1-5任意一项所述的系统，其特征在于，所述的生物为真核生物、原核生物或具有DNA的非细胞生物。
  7. 根据权利要求1-6任意一项所述的系统，其特征在于，所述的基因突变类型包括插入型和替换型基因突变。
  8. 根据权利要求7所述的系统，其特征在于，所述的插入型和替换型基因突变是指在基因的编码区产生功能缺失或功能获得型突变，或者在基因的调控区产生功能缺失或功能获得型突变。
  9. 一种应用权利要求1-8任意一项所述系统在生物基因组上创制基因突变的方法。
  10. 根据权利要求1-8任意一项所述系统或权利要求9所述方法，其特征在于，所述系统在酵母基因组的ILV2基因上创制了的功能获得型基因突变包括K251F、M354L、L589F、F590S、F590V和F590L中的一个或多个突变，优选地，包括K251F、M354L、F590S、F590V和L589F/F590L。
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