CN107988229A - 一种利用CRISPR-Cas修饰OsTAC1基因获得分蘖改变的水稻的方法 - Google Patents
一种利用CRISPR-Cas修饰OsTAC1基因获得分蘖改变的水稻的方法 Download PDFInfo
- Publication number
- CN107988229A CN107988229A CN201810010122.7A CN201810010122A CN107988229A CN 107988229 A CN107988229 A CN 107988229A CN 201810010122 A CN201810010122 A CN 201810010122A CN 107988229 A CN107988229 A CN 107988229A
- Authority
- CN
- China
- Prior art keywords
- sgrna
- rice
- tac1
- ostac1
- tillering angle
- 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.)
- Granted
Links
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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明提供的培育减小分蘖角度转基因植物的方法,即编辑了一种调控水稻分蘖角度的蛋白质,来源于稻属水稻(Oryza sativa var.93‑11),名称为OsTAC1。具体是将上述的编码基因利用CRISPR/Cas9基因编辑技术对OsTAC1进行敲除从而得到转基因植物,所述转基因植物的分蘖角度相比于对照野生型植株变小。另外本发明阐释了功能弱化型突变和功能丧失型突变与植株分蘖角度之间的关系,本发明提供的sgRNA具有较高的编辑效率,具有较好的应用前景。
Description
技术领域
本发明涉及基因工程领域,具体涉及利用CRISPR/Cas9系统编辑OsTAC1基因,进而获得分蘖角度减小的水稻品种。
背景技术
水稻(Oryza sativa L.)作为我国乃至世界最重要的粮食作物之一,随着人口数量的不断增加,其产量的提高对解决未来全球粮食问题具有十分重要的战略意义。
株型是影响水稻群体产量的重要农艺性状之一,提高水稻的种植密度是增加水稻产量的有效方法。水稻的分蘖角度是指侧生分蘖和主茎之间的夹角,作为影响水稻理想株型的重要因子之一,它决定植株的单位面积种植密度及作物产量。理想的分蘖角度既能避免角度过小导致田间湿度过高而诱发植物病害,又能避免因匍匐生长导致的光合作用效率降低和单位面积种植密度降低导致的产量下降,因此对水稻株型进行精确的人工改良具有重要意义。
传统的株型育种一般包括系统育种、杂交育种和传统基因工程育种。水稻系统育种选育新品种是由一个自然变异株个体发展形成的一个新系统。其优点是直接利用自然变异,省去人工创造变异环节,选育的优良个体一般较纯合,性状易稳定,能较快地育成新品种;缺点是仅能改良已有品种,不能根据需求创造新的类型,难以育成突破性品种。水稻杂交育种是将具有不同遗传背景地品种进行杂交,经过选择培育获得新品种的方法。优点是可将两个或两个以上的亲本的优良基因相融合,通过后代基因分离、重组,产生新的优良品种,借助于杂种优势,可育成突破性的新品种,育种效果显著;缺点是选育周期长,性状不稳定易发生分离。传统基因工程育种是利用转基因技术将决定某个特定品种遗传属性的目标基因转移到植物体内,改造植物基因组,实现基因重组并培育出高产、多抗、优质的新品种。优点是:克服了传统育种周期长、基因转移不可控的问题,可实现基因的定性定向高效聚合;缺点是无法实现基因的定点敲除、外源基因的表达存在不可控现象和转基因的安全性问题尚未定论。目前针对水稻分蘖的研究主要体现在增加水稻分蘖的研究上。比如,CN1844396A中公开了一种调控水稻分蘖角度的方法,是将调控水稻分蘖角度的基因导入水稻组织或细胞,水稻分蘖角度获得调控。CN 106518993 A通过降低OsAAP3基因表达,可以使正常的水稻分蘖数和每株穗数增加,因此OsAAP3基因可用于水稻选育中以提高水稻产量。OsAAP3基因在阐述氨基酸运输影响植物生长及发育过程方面具有重要的应用价值。都没有涉及针对降低分蘖效果的基因研究。
CRISPR/Cas是一种具有核酸内切酶活性的复合体,识别特定的DNA序列,进行特定位点切割造成双链DNA断裂,在没有模板的条件下,发生非同源重组末端连接,造成移码突变,导致基因敲除。这一技术由于能快速、简便、高效地靶向基因组任何基因,从而引起了广泛的关注,在2012年开始像爆炸一般流行开来。由于其容易操作、可以同时靶向多个基因,可以高通量制备、造价低等优势,Cas9已经成为一种发展最快的技术。正是由于其优越性,这一技术在Nature推荐的2013十大进展中位列第一,在Science推荐的2013十大进展中位列第二位。Cas9靶向切割DNA是通过两种小RNA--crRNA(CRISPR RNA)和tracrRNA(transactivating crRNA)和靶序列互补识别的原理实现的。现在已经把两种小RNA融合成一条RNA链,简称sgRNA(single guide RNA)。因此,sgRNA能否做到特异性、精确靶向目标基因是CRISPR/Cas9能否特异性敲除目标基因的先决条件,无论是脱靶还是错误靶向,都会影响CRISPR/Cas9对目标基因的特异性敲除。因此,能够设计、制备出精确性和特异性靶向目标基因的sgRNA成为CRISPR/Cas9基因敲除的关键技术。
目前随着CRISPR/Cas9基因编辑技术的发展完善,可以在基因组的特定位点产DNA双链断裂,实现基因敲除、定点插入或替换,可以定向的对水稻基因组进行编辑,并且通过后代的分离可已将含有转基因成分的植株分离出去,达到精确快速改良品种的目的。
发明内容
本发明的目的在于解决现有技术中存在的问题,提供一种改良植物分蘖角度的新方法。
本发明的目的通过下述技术方案实现:
本发明所编辑的与植物分蘖角度相关的基因,名称为OsTAC1(Tiller anglecontrol 1)。OsTAC1是一个在水稻育种实践中被广泛应用的株型功能基因,与籼稻相比,粳稻中的OsTAC1基因位于3’端非编码区的第四个内含子的剪切位点发生由“agga”突变为“ggga”导致其表达量下调,从而导致分蘖角度减小。OsTAC1由259个氨基酸残基组成(如序列1所示)。
本发明选用散生籼稻品种93-11(Oryza sativa L.93-11,中国农业科学院作物科学研究所,国家种质库)为研究品种,利用CRISPR/Cas9技术对93-11基因组上的OsTAC1基因进行编辑。分别在OsTAC1基因第2,3和4个外显子处设计4个sgRNA位点,分别记为sgRNA-exon2、sgRNA-exon3-1、sgRNA-exon3-2和sgRNA-exon4(序列如下)(所述序列均是发明人通过创造性劳动初筛得到的,非通过常规软件设计简单获得)。通过CRISPR/Cas9蛋白的定点切割与随机性修复,产生了不同的编辑类型。
(下划线所示序列为20bp的sgRNA,加粗碱基为PAM位点)
本发明的另一个目的是提供一种减小植株分蘖角度的转基因方法。
本发明提供的培育减小分蘖角度转基因植物的方法,具体是将上述的编码基因利用CRISPR/Cas9基因编辑技术进行敲除从而得到转基因植物,所述转基因植物的分蘖角度相比于对照野生型植株变小。
本发明的实验证明,将提供OsTAC1的编码基因在93-11中敲除后获得的转基因植株,与未敲除该基因的野生型植株相比,分蘖角度小于野生型植株,因此,该基因可应用于植物株型遗传改良等工作。
本研究所设计的sgRNA位点、CRISPR/Cas9编辑产生的不同编辑类型以及突变体分蘖角度的表型特征均属于本发明的保护范围。
本研究阐述的功能弱化型突变和功能丧失型突变这两种突变类型所属含义以及其与分蘖角度之间的对应关系属于本发明的保护范围。
含有所述编码基因的重组载体、重组菌、转基因细胞系或表达盒均属于本发明的保护范围。
附图说明
图1表型结果对照图;左边图为野生型和4个基因编辑后的水稻表型图,右边二幅分别为分蘖角度和分蘖个数对比图。
图2 4个sgRNA位点的编辑结果图。
图3 tac1-sgRNA-exon3-1编辑类型和植株表型图。
具体实施方式
下述实施例中所使用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、试剂等,如无特殊说明,均可从商业途径得到。
举例说明本发明的具体实施过程,使本领域技术人员按照其不需要创造性劳动就能完成该发明即可,实施例的限定不能作为限定发明人保护范围的局限。
实施例1表达载体的构建
根据被AarI切割后的pCas9载体骨架序列,将得到的20bp前后加接头序列AGATGATCCGTGGCA…N20…GTTTTAGAGCTATGC作为F引物,将所得到的F引物进行反向互补,得到R引物,送交公司合成(序列如下)。
AGATGATCCGTGGCATGCACCATCAATGAGAACAAGTTTTAGAGCTATGC sgRNA-exon2-F
GCATAGCTCTAAAACTTGTTCTCATTGATGGTGCATGCCACGGATCATCT sgRNA-exon2-R
AGATGATCCGTGGCAATACTTGCAATTGGCACGCTGTTTTAGAGCTATGC sgRNA-exon3-1-F
GCATAGCTCTAAAACAGCGTGCCAATTGCAAGTATTGCCACGGATCATCT sgRNA-exon3-1-R
AGATGATCCGTGGCACGAAAATCGTCATTGTTGCTGTTTTAGAGCTATGC sgRNA-exon3-1-F
GCATAGCTCTAAAACAGCAACAATGACGATTTTCGTGCCACGGATCATCT sgRNA-exon3-1-R
AGATGATCCGTGGCATGTAAAATAAGTAGGTCATGGTTTTAGAGCTATGC sgRNA-exon4-F
GCATAGCTCTAAAACCATGACCTACTTATTTTACATGCCACGGATCATCT sgRNA-exon4-R
(下划线所示序列为20bp的sgRNA)
在10ul体系中各加入1ul的F及R引物,其余用水补齐。94℃10min,0.1℃/s退火至15℃,15℃保持10min,完成退火。
将pCas9载体用AarI进行酶切处理,胶回收15652bp大小的目标片段,取1ul退火产物与酶切后的pCas9载体进行infusion连接,得到重组植物表达载体,转入DH5α,涂于Spec固体培养基上。挑取阳性单克隆,提取质粒测序验证,将测序正确含有sgRNA序列的重组载体分别命名为tac1-sgRNA-exon2、tac1-sgRNA-exon3-1、tac1-sgRNA-exon3-2和tac1-sgRNA-exon4。
用热激法将tac1-sgRNA-exon2、tac1-sgRNA-exon3-1、tac1-sgRNA-exon3-2和tac1-sgRNA-exon4转化农杆菌为EHA105菌株得到重组体,提取质粒进行测序验证,将测序验证正确的重组菌株分别命名EH-tac1-sgRNA-exon2、EH-tac1-sgRNA-exon3-1、EH-tac1-sgRNA-exon3-2和EH-tac1-sgRNA-exon4。
实施例2农杆菌介导转化
以EH-tac1-sgRNA-exon3-1为例。利用农杆菌介导将构建好的EH-tac1-sgRNA-exon3-1转化籼稻品种93-11(Oryza sativa L.93-11,中国农业科学院作物科学研究所,国家种质库)具体方法为:
1)28℃培养EH-tac1-sgRNA-exon3-1菌液16小时,收集菌体,并稀释到含有100μmol/L的N6液体培养基(Sigma公司购买,C1416),至浓度为OD600≈0.8,获得菌液;
2)将培养至一个月的93-11成熟胚胚芽鞘组织与上述菌液混合侵染30min,滤纸吸干菌液后转入共培养培养基(N6固体共培养培养基,Sigma公司购买)中,24℃共培养3天;
3)将上述愈伤组织接种在含有150mg/L潮霉素(Sigma公司购买)的N6固体筛选培养基上第一次筛选16天;
4)挑取健康愈伤组织转入200mg/L潮霉素的N6固体筛选培养基上第二次筛选,每15天继代一次;
5)挑取抗性愈伤转入含有150mg/L潮霉素的分化培养基(Sigma公司购买,M519,M524)上分化;
6)分化成苗的再生水稻植株即为所获得的T0代转基因水稻,共获得4株T0代转tac1-exon3-1水稻。
采用同样的方法将空载体pCas9转入农杆菌EHA105获得重组菌EH-pCas9,再采用上述方法将重组菌EH-pCas9导入水稻品种93-11中得到3株T0代转pCas9水稻。
实施例3转基因水稻分子鉴定
提取上述获得4株T0代转tac1-sgRNA-exon3-1水稻的DNA作为模板进行PCR分子检测,以T0代转pCas9水稻为对照。具体方法为:以primer3-1-F:ACCAGGTGTTCAATTGGCTG和primer3-1-R:CCCCAGCAACAATGACGATT为引物对进行PCR扩增,PCR反应体系:10×PCRBuffer for KOD-plus-Neo 5ul,2mM dNTPs 5ul,25mM MgSO4 3ul,DNA(200ng/ul)1ul,primer3-1-F(10pmol/ul)1.5ul,primer3-1-R(10pmol/ul)1.5ul,KOD-plus-Neo(1U/ul)1ul,ddH2O 32ul,总体积50ul。扩增反应体系:94℃2min;98℃10sec,58℃30sec,68℃30min,40个循环,PCR产物送公司测序验证。结果表明,4株T0转tac1-sgRNA-exon3-1植株全为被编辑的阳性植株。
依上所述方法,得到转基因植株数目如表1:
从表4的结果可以看出,四个sgRNA均可以实现tac1的特异性突变效果,其中尤其以tac1-sgRNA-exon3-1和tac1-sgRNA-exon3-2可以实现100%的编辑效率,其重组效率超出了本领域常规的范围。这也充分说明,在前期申请人付出创造性劳动筛选得到的这四种sgRNA具有本领域常规方法所无以比拟的优势,具有较强的编辑效率。
实施例4转基因水稻表型鉴定
将从阳性T0代转基因植株收获的T1代种子种于大田进行表型分析,以转野生型水稻(93-11)为对照。表型结果如图1所示,4个sgRNA位点的编辑结果如图2所示。从图1可以看出,四个转基因植物与野生型对照相比,分蘖数基本保持不变,而在分蘖数相差不多的情况下,4个sgRNA位点均得到tac1突变植株,获得的转基因植株较对照植株分蘖角度明显变小。
实施例5梯度表型的发掘
在T1代tac1突变体植株中,存在两种编辑类型,即,功能弱化型突变(丢失或插入3n(n≥1)个碱基)和功能丧失型突变(丢失或插入非3倍数的碱基),这两种突变类型会显现出不同的分蘖角度表型。
在转tac1-sgRNA-exon3-1的T1代植株中,存在tac1-1(缺失21bp碱基,即功能弱化型突变)和tac1-2(缺失8bp碱基,即功能丧失型突变)两种突变类型,其中tac1-1缺失了7个氨基酸(序列如8),而tac1-2引发移码导致编码蛋白提前终止(序列如9)。结果显示功能弱化突变体tac1-1的分蘖角度介于野生型和功能丧失突变体tac1-2之间,证明功能弱化突变和功能丧失突变可产生不同梯度的分蘖角度表型。编辑类型和植株表型如图3所示。但是二者均可以实现对水稻的分蘖角度进行改变的作用。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
序列表
<120> 一种利用CRISPR-Cas 修饰OsTAC1 基因获得分蘖改变的水稻的方法
<141> 2018-01-05
<160> 0
<170> SIPOSequenceListing 1.0
Claims (10)
1.一种sgRNA,其用于基因编辑权利要求2所述的OsTAC1基因。
2.一种sgRNA,其序列如SEQ ID NO:4-7任一所示。
3.一种减小植株分蘖角度的转基因方法,具体是使用权利要求2所述的sgRNA进行CRISPR/Cas9基因编辑进行基因敲除,从而得到转基因植物,所述转基因植物的分蘖角度相比于对照野生型植株变小。
4.根据权利要求3所述的方法,其中植物为水稻。
5.一种利用CRISPR/Cas9技术对籼稻散生品种93-11进行基因编辑的方法,以籼稻品种93-11的OsTAC1基因为靶点,在基因第2,3和4个外显子处设计4个sgRNA位点,分别记为sgRNA-exon2、sgRNA-exon3-1、sgRNA-exon3-2和sgRNA-exon4,序列如下,将sgRNA与cas9一起倒入到水稻中,通过CRISPR/Cas9蛋白的定点切割与随机性修复,制备得到基因编辑水稻;其中
sgRNA-exon2:TGCACCATCAATGAGAACAA;
sgRNA-exon3-1:ATACTTGCAATTGGCACGCT;
sgRNA-exon3-2:CGAAAATCGTCATTGTTGCT;
sgRNA-exon4:TGTAAAATAAGTAGGTCATG。
6.水稻OsTAC1基因敲除在制备水稻分蘖角度减小的转基因水稻中的应用。
7.如权利要求6所述的应用,其中OsTAC1基因的序列如SEQ ID NO:2所示。
8.如权利要求6或7所述的应用,其中水稻为籼稻散生品种93-11。
9.SEQ ID NO:8所示的tac1-1功能弱化型突变在减小水稻分蘖角度中的应用。
10.SEQ ID NO:9所示的tac1-2功能丧失型突变在减小水稻分蘖角度中的应用;其中SEQ ID NO:8所示的功能弱化突变体tac1-1的分蘖角度介于野生型和SEQ ID NO:9所示的功能丧失突变体tac1-2之间,证明功能弱化突变和功能丧失突变可产生不同梯度的分蘖角度表型。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810010122.7A CN107988229B (zh) | 2018-01-05 | 2018-01-05 | 一种利用CRISPR-Cas修饰OsTAC1基因获得分蘖改变的水稻的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810010122.7A CN107988229B (zh) | 2018-01-05 | 2018-01-05 | 一种利用CRISPR-Cas修饰OsTAC1基因获得分蘖改变的水稻的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107988229A true CN107988229A (zh) | 2018-05-04 |
CN107988229B CN107988229B (zh) | 2020-01-07 |
Family
ID=62040673
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810010122.7A Active CN107988229B (zh) | 2018-01-05 | 2018-01-05 | 一种利用CRISPR-Cas修饰OsTAC1基因获得分蘖改变的水稻的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107988229B (zh) |
Cited By (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
US11999947B2 (en) | 2023-02-24 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1844396A (zh) * | 2006-04-28 | 2006-10-11 | 中国农业大学 | 调控水稻分蘖角度的基因及其编码蛋白与应用 |
CN104805084A (zh) * | 2014-01-27 | 2015-07-29 | 中国科学院遗传与发育生物学研究所 | 水稻叶夹角的表观调控位点及其应用 |
-
2018
- 2018-01-05 CN CN201810010122.7A patent/CN107988229B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1844396A (zh) * | 2006-04-28 | 2006-10-11 | 中国农业大学 | 调控水稻分蘖角度的基因及其编码蛋白与应用 |
CN104805084A (zh) * | 2014-01-27 | 2015-07-29 | 中国科学院遗传与发育生物学研究所 | 水稻叶夹角的表观调控位点及其应用 |
Non-Patent Citations (2)
Title |
---|
BAISHENG YU ET AL.: "TAC1, a major quantitative trait locus controlling tiller angle in rice", 《THE PLANT JOURNAL》 * |
余敏祥: "利用CRISPR/Cas系统构建水稻OsGRF基因敲除株系", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
Cited By (37)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US12006520B2 (en) | 2019-06-14 | 2024-06-11 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
US11999947B2 (en) | 2023-02-24 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
CN107988229B (zh) | 2020-01-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107988229A (zh) | 一种利用CRISPR-Cas修饰OsTAC1基因获得分蘖改变的水稻的方法 | |
CN106544357B (zh) | 一种培育镉低积累籼稻品种的方法 | |
CN106191107B (zh) | 一种降低水稻籽粒落粒性的分子改良方法 | |
CN107129993B (zh) | 一种修饰的抗草甘膦基因及抗草甘膦水稻的培育方法 | |
CN108103092A (zh) | 利用CRISPR-Cas系统修饰OsHPH基因获得矮化水稻的系统及其应用 | |
CN111996209B (zh) | 孤雌生殖单倍体诱导基因dmp及其应用 | |
CN112063626B (zh) | 玉米基因ZmRAVL1和功能位点及其用途 | |
CN102634522B (zh) | 控制水稻育性的基因及其编码蛋白和应用 | |
CN107475210A (zh) | 一种水稻白叶枯病抗性相关基因OsABA2及其应用 | |
CN109207509B (zh) | 一种定向、高效培育耐盐水稻品种的育种方法 | |
CN110881367A (zh) | 一种玉米事件t抗-4及其使用方法 | |
CN107805632A (zh) | OsMKK6蛋白及编码基因在调控植物种子发育中的应用 | |
Kang et al. | A robust genome-editing method for wild plant species Nicotiana attenuata | |
CN112522291A (zh) | 水稻OsSH3P2基因及其应用 | |
CN116574731A (zh) | 一种用于白桦CRIPSR/Cas9基因编辑的启动子及其应用 | |
CN107338231A (zh) | OsMPK21-1蛋白及其编码基因在调控植物抗旱性中的应用 | |
CN100372935C (zh) | 辣椒抗根结线虫基因的克隆及其应用 | |
CN103320463B (zh) | 用RNAi技术控制水稻育性基因获得水稻不育系的方法 | |
CN114591984B (zh) | OsAP79基因诱导水稻抗褐飞虱的应用 | |
CN116926109B (zh) | 一种植物程序化花粉自清除CRISPR/Cas基因编辑方法 | |
CN117305326B (zh) | 青花菜BoCENH3基因及其在单倍体诱导中的应用 | |
CN116789785B (zh) | 长雄蕊野生稻高产、高光效基因FarL1及其应用 | |
CN106086063A (zh) | 一种基于同尾酶构建的RNAi载体及其应用 | |
CN116063423A (zh) | 一种提高单倍体诱导效率的突变型OsPLA1m1及其应用 | |
CN105420207B (zh) | 与水稻叶绿体rna聚合酶pep及叶绿体发育相关的蛋白及其编码基因和应用 |
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 |