CN105779448A - 一种棉花启动子GbU6-7PS及应用 - Google Patents
一种棉花启动子GbU6-7PS及应用 Download PDFInfo
- Publication number
- CN105779448A CN105779448A CN201510885400.XA CN201510885400A CN105779448A CN 105779448 A CN105779448 A CN 105779448A CN 201510885400 A CN201510885400 A CN 201510885400A CN 105779448 A CN105779448 A CN 105779448A
- Authority
- CN
- China
- Prior art keywords
- gbu6
- promoter
- cotton
- gus
- expression vector
- 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
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种棉花启动子GbU6‑7PS及应用,该启动子的核苷酸序列如SEQ ID NO.1所示,其互补链的核苷酸序列如SEQ ID NO.2所示;本发明棉花启动子GbU6‑7PS通过进行第一轮PCR扩增获得覆盖全长GbU6‑7启动子序列的片段,得到GbU6‑7P;再通过第二轮PCR扩增,以GbU6‑7P为供体质粒模板,以GbU6‑5P::GUS为受体质粒模板,采用Transfer PCR方法对GbU6‑7P启动子进行截短克隆后得到。本发明的棉花启动子GbU6‑7PS不仅适合用于棉花,而且片段很短,长度只有190bp,符合构建CRISPR/Cas9基因组编辑载体的要求,能显著提高sgRNA的转录水平,进而可能提高基因组编辑效率。
Description
技术领域
本发明属于生物技术领域,尤其涉及一种棉花启动子GbU6-7PS及应用。
背景技术
基因组编辑技术是一种研究基因功能的重要工具,它能精确修饰受体细胞染色体特定位点的基因,可以高效产生特定基因的功能失活突变体,能为生物功能基因组研究提供优质的遗传材料。所以该技术自诞生以来,就受到广大生物学家的青睐。II型CRISPR/Cas9基因组编辑系统是继锌指核酸酶(ZFNs)和TALE核酸酶(TALENs)之后,另一种对基因组进行高效定点修饰的新技术。sgRNA和Cas9是该技术系统的两个必要元件,其中sgRNA在CRISPR/Cas9基因组编辑过程中起到靶向结合目的基因位点的功能。在CRISPR/Cas9技术系统中,sgRNA的转录通常由U6启动子驱动完成,因为其转录活性相对较高,而且都有着明确的转录起始位点,精确起始转录具有靶向功能的sgRNA能消除无关DNA序列的转录,从而大大减少脱靶效应的产生。
U6启动子虽然具有高效转录的特点,但是它在同源关系较远的不同物种之间不一定适用,而且同一物种基因组中存在多个U6启动子,这些启动子的转录效率也不相同。另外由于Cas9基因巨大(大于4 000bp),而且驱动Cas9基因表达的35S启动子长度也在1 000bp左右,这两个片段上分布着绝大多数常用的限制性内切酶位点。因此在构建sgRNA和Cas9共表达载体时,可用于sgRNA重组片段连接的酶切位点非常少。所以这就要求U6启动子上不能再出现这些酶切位点,而只有更短的片段才有可能满足这个要求。因此克隆的U6启动子片段越短越便于CRISPR/Cas9载体的构建。
棉花是我国重要的经济作物和纺织工业原料。其基因组中至少有10种U6启动子。克隆适用于棉花CRISPR/Cas9基因组编辑技术系统的、高转录效率的、短片段的U6启动子,对于利用CRISPR/Cas9基因组编辑技术进行棉花分子育种具有重要意义。
发明内容
本发明的目的在于提供一种棉花启动子GbU6-7PS及其制备方法。
本发明的再一目的在于提供上述棉花启动子GbU6-7PS的应用。
本发明的再一目的在于提供包含上述棉花启动子GbU6-7PS的表达载体及应用。
本发明是这样实现的,一种棉花启动子GbU6-7PS,该启动子核苷酸序列如SEQ IDNO.1所示,该启动子互补链的核苷酸序列如SEQ ID NO.2所示。本发明的棉花启动子GbU6-7PS由190个核苷酸组成,含有USE和TATA作用元件。
或者在严格条件下与上述SEQ ID NO.1所示核苷酸序列或SEQ ID NO.2所示核苷酸序列杂交且具有启动子功能的DNA分子;
或者与上述SEQ ID NO.1所示核苷酸序列或SEQ ID NO.2所示核苷酸序列具有90%以上同源性,且具有启动子功能的DNA分子。
优选的,所述严格条件为:在0.1×SSPE(或0.1×SSC)、0.1%SDS的溶液中,65℃条件下杂交并洗膜。
本发明进一步提供了上述棉花启动子GbU6-7PS的制备方法,该方法包括以下步骤:
第一步进行第一轮PCR扩增获得覆盖全长GbU6-7启动子序列的片段,第一轮PCR的程序为:94℃3min;98℃30s,60℃30s,72℃60s,35个循环,然后72℃10min;最后克隆测序验证,正确的克隆质粒被命名为GbU6-7P;
所用引物:GbU6-7PF:ACCTTTATGAATGCCGAGTAT;
GbU6-7PR:ACAAACCACAGGGTTATGAG;
第二步进行第二轮PCR扩增,以GbU6-7P为供体质粒模板,以GbU6-5P::GUS为受体质粒模板,采用Transfer PCR方法对GbU6-7P启动子进行截短克隆,获得的截短启动子被命名为GbU6-7PS,其中,Transfer PCR的程序为:95℃1min;95℃30s,60℃30s,72℃40s,13个循环;95℃30s,67℃1min,72℃4min,20个循环;72℃8min;
所用引物:
T-GbU6-7PS F:CCGCCAGTGTGCTGGAATTGCCCTTAATCCCACTCTACAGCCATAAG;
T-GbU6-7PS R:GGGTTTCTACAGGACGTAACATGAAGACCCATTACCTGCTAGCTGCTTCTT。
本发明进一步提供了上述棉花启动子GbU6-7PS在驱动GUS基因转录方面的应用。
优选的,所述棉花启动子GbU6-7PS的重组载体、表达盒、转基因细胞系或重组菌在驱动GUS基因转录方面的应用。
优选的,所述棉花启动子GbU6-7PS驱动GUS基因在棉花花粉中的高效转录。
本发明进一步提供了一种GUS融合表达载体,该表达载体是通过将所述棉花启动子GbU6-7PS与GUS基因连接后,克隆至植物表达载体制备得到。
优选的,所述植物表达载体为pCAMBIA 1300。
本发明进一步提供了上述GUS融合表达载体在驱动GUS基因转录方面的应用。
U6启动子是CRISPR/Cas9基因组编辑载体系统中驱动sgRNA转录的重要元件。本发明提供了一种棉花启动子GbU6-7PS及应用。在本发明中,将已经克隆获得的海岛棉GbU6-7P启动子(长度为1395bp)(参考文献:雷建峰,伍娟,陈晓俊,於添平,倪志勇,李月,张巨松,刘晓东.棉花花粉中高效转录U6启动子的克隆及功能分析.中国农业科学,2015,48(19):3794-3802.),采用Transfer PCR方法成功地截短出190bp长的棉花启动子GbU6-7PS,并构建启动子驱动的GUS融合表达载体GbU6-7PS::GUS-P1300。将构建好的GbU6-7PS::GUS-P1300与阳性对照CaMV35S::GUS-P1300植物表达载体一起利用农杆菌真空渗透转化法分别转化棉花花粉。经GUS组织化学染色显示:克隆得到的190bp的棉花启动子GbU6-7PS能驱动GUS基因在棉花花粉中转录,棉花花粉被染成显著深蓝色。
相比于现有技术的缺点和不足,本发明具有以下有益效果:本发明的棉花启动子GbU6-7PS不仅适合用于棉花,而且片段很短,长度只有190bp,符合构建CRISPR/Cas9基因组编辑载体的要求,能显著提高sgRNA的转录水平,进而可能提高基因组编辑效率。
附图说明
图1是本发明实施例中GbU6-7P启动子的第一轮PCR扩增结果图;
图2是本发明实施例中棉花启动子GbU6-7PS的Transfer PCR扩增结果图;
图3是本发明实施例中棉花启动子GbU6-7PS驱动GUS在棉花花粉中的瞬时表达结果图,参见附图3A所示,图B、C分别是阳性对照CaMV35S强启动子驱动GUS基因表达和阴性对照pCAMBIA 1300转化花粉染色的结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
1、试验材料
试验所用GbU6-5P::GUS(参考文献:雷建峰,伍娟,陈晓俊,於添平,倪志勇,李月,张巨松,刘晓东.棉花花粉中高效转录U6启动子的克隆及功能分析.中国农业科学,2015,48(19):3794-3802.)、农杆菌GV3101、棉花品种新海16均为新疆农业大学农业生物技术重点实验室保存。
各种限制性内切酶购于Fermentas公司;T4 DNA连接酶、Blunt Zero平端载体、Trans1-T1感受态细胞、胶回收试剂盒、Taq DNA Polymerase、RNase A、KD Plus DNAPolymerase、1Kb Plus DNA Ladder均购自于北京全式金生物技术有限公司;Phusion超保真DNA聚合酶购自于纽英伦生物技术(北京)有限公司;其它常规试剂均为国产分析纯;引物合成及测序均由上海杰李生物技术有限公司完成。
2、GbU6-7PS启动子的克隆
(1)第一轮PCR
通过PCR扩增(参考文献:雷建峰,伍娟,陈晓俊,於添平,倪志勇,李月,张巨松,刘晓东.棉花花粉中高效转录U6启动子的克隆及功能分析.中国农业科学,2015,48(19):3794-3802.)。获得棉花品种新海16覆盖全长GbU6-7启动子序列的片段,PCR的程序为:94℃3min;98℃30s,60℃30s,72℃60s,35个循环,然后72℃10min;最后克隆测序验证,正确的克隆质粒被命名为GbU6-7P;所用引物为GbU6-7P F:ACCTTTATGAATGCCGAGTAT;GbU6-7P R:ACAAACCACAGGGTTATGAG。克隆结果如图1所示。
(2)第二轮PCR
本发明第二轮PCR是精确克隆截短的棉花启动子GbU6-7PS,以第一轮PCR克隆的GbU6-7P启动子质粒为供体质粒模板,以拟南芥GbU6-5P::GUS的质粒为受体质粒模板进行由两轮PCR组成的TransferPCR(参考文献:Erijman A,Shifman J M,Peleg Y.A single-tube assembly of DNA using the transfer-PCR(TPCR)platform.Methods inMolecular Biology,2014,1116:89-101.),对GbU6-7P启动子进行截短。
根据已克隆的GbU6-7P启动子序列和GbU6-5P::GUS载体序列设计棉花启动子GbU6-7PS的Transfer PCR引物:
T-GbU6-7PS F:CCGCCAGTGTGCTGGAATTGCCCTTAATCCCACTCTACAGCCATAAG;
T-GbU6-7PS R:GGGTTTCTACAGGACGTAACATGAAGACCCATTACCTGCTAGCTGCTTCTT。
以GbU6-7P为供体质粒模板,以GbU6-5P::GUS为受体质粒模板,采用Transfer PCR方法对GbU6-7P启动子进行截短克隆,同时完成GbU6-7P启动子与GUS基因融合载体的构建,获得的截短启动子被命名为GbU6-7PS,其中,Transfer PCR的程序为:95℃1min;95℃30s,60℃1min,72℃40s,13个循环;95℃30s,67℃1min,72℃4min,20个循环;72℃8min;PCR产物电泳结果如图2所示。
取酶切产物10μL转化Trans1-T1感受态细胞。对初步获得的截短的质粒采用BamHⅠ和Hind Ⅲ进行酶切鉴定。以上截短的启动子质粒经BamH Ⅰ和Hind Ⅲ酶切鉴定与预期目标片段大小相符(图略)。
酶切正确的质粒进行测序验证,经测序比对为预期的截短棉花启动子GbU6-7PS,棉花启动子GbU6-7PS核苷酸序列如SEQ ID NO.1所示,该启动子互补链的核苷酸序列如SEQID NO.2所示测定序列。测序正确的质粒被命名为GbU6-7PS::GUS
3、GUS融合表达载体GbU6-7PS::GUS-P1300的构建
用HindⅢ和BamH Ⅰ双酶切植物表达载体pCAMBIA 1300和构建好的GbU6-7PS::GUS的融合表达载体,分别回收目标片段,T4 DNA连接酶4℃连接过夜。转化,提质粒。
再用HindⅢ和BamH Ⅰ双酶切鉴定阳性克隆,将酶切鉴定正确的质粒命名为:GbU6-7PS::GUS-P1300质粒。
经酶切鉴定正确后,转入农杆菌感受态GV3101,28℃倒置培养两天,挑取阳性克隆于LB培养基(含50μg·mL-1kan和25μg·mL-1Rif)培养至对数生长期,用于下一步侵染。
4、农杆菌真空渗透转化棉花花粉
将构建好的GbU6-7PS::GUS-P1300以及阳性对照CaMV35S::GUS和阴性对照pCAMBIA1300空载体的农杆菌按1:100比例接种于LB培养基(含50g·mL-1kan和25g·mL- 1Rif)中活化。28℃,180rpm摇菌,摇至菌液的OD600=0.6-1.2时,分别吸取不同体积的活化菌液接种到6mL LB培养基(含50μg·mL-1kan和25μg·mL-1Rif)中,使得每个处理起始OD值相同,再次28℃,180rpm摇菌,统一摇至菌液的OD600=1.6时,12000rpm离心5min收集菌体,弃上清液,重悬于花粉萌发培养基(参考文献:张燕红,黄乐平,周小云,王冬梅.农杆菌真空渗透法转化棉花花粉的初步研究.棉花学报,2008,05:354-358.)中(0.1%H3BO3、0.3%Ca(NO3)2、0.2%MgSO4·7H2O、0.1%KNO3、45%蔗糖),收集新鲜棉花花粉,等量分成多份。每份各加入上述不同的农杆菌重悬液,并在-0.05MPa压力下抽真空30min。每种农杆菌转化进行技术重复三次,生物学重复两次。
5、GbU6-7PS::GUS在棉花花粉中的瞬时表达分析
将抽完真空后的棉花花粉悬浮液100rpm离心1min,弃上清液,收集转化后的花粉,并用蒸馏水漂洗4-5次,除去农杆菌。加入适量的GUS染液(0.5mol·L-1磷酸缓冲液,pH 7.0;0.5mol·L-1EDTA,pH 8.0;10%Triton X-100;20mmol·L-1X-Gluc),37℃,180rpm震荡染色3~4h,100rpm离心5min,吸取上清GUS染液保留备用。用蒸馏水漂洗转化后的花粉4~5次,100rpm离心5min确保去除残留的GUS染液。收集的花粉在体式显微镜下观察拍照。同时取处理转化后GUS染液的蓝色上清液在620nm波长(蓝色上清液的最大吸收波长)处测定光吸收值。
通过GUS组织化学染色发现:截短的棉花启动子GbU6-7PS能驱动GUS基因的表达,棉花花粉被染成蓝色且染色相对较深,接近于阳性对照,而阴性对照pCAMBIA1300转化的花粉未被染蓝色(如图3所示)。
6、结论
本发明的棉花启动子GbU6-7PS不仅适合用于棉花,而且片段很短,长度只有190bp,符合构建CRISPR/Cas9基因组编辑载体的要求,能显著提高sgRNA的转录水平,进而可能提高基因组编辑效率。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种棉花启动子GbU6-7PS,其特征在于,该启动子的核苷酸序列如SEQ ID NO.1所示,该启动子的互补链的核苷酸序列如SEQ ID NO.2所示。
2.权利要求1所述的棉花启动子GbU6-7PS的制备方法,其特征在于,该方法包括以下步骤:
第一步进行第一轮PCR扩增获得覆盖全长GbU6-7启动子序列的片段,第一轮PCR的程序为:94℃3min;98℃30s,60℃30s,72℃60s,35个循环,然后72℃10min;最后克隆测序验证,正确的克隆质粒被命名为GbU6-7P;
所用引物:GbU6-7PF:ACCTTTATGAATGCCGAGTAT;
GbU6-7PR:ACAAACCACAGGGTTATGAG。
第二步进行第二轮PCR扩增,以GbU6-7P为供体质粒模板,以GbU6-5P::GUS为受体质粒模板,采用Transfer PCR方法对GbU6-7P启动子进行截短克隆,获得的截短启动子被命名为GbU6-7PS,其中,Transfer PCR的程序为:95℃1min;95℃30s,60℃30s,72℃40s,13个循环;95℃30s,67℃1min,72℃4min,20个循环;72℃8min;
所用引物:
T-GbU6-7PS F:CCGCCAGTGTGCTGGAATTGCCCTTAATCCCACTCTACAGCCATAAG;
T-GbU6-7PS R:GGGTTTCTACAGGACGTAACATGAAGACCCATTACCTGCTAGCTGCTTCTT。
3.权利要求1所述的棉花启动子GbU6-7PS在驱动GUS基因转录方面的应用。
4.如权利要求3所述的应用,其特征在于,所述棉花启动子GbU6-7PS的重组载体、表达盒、转基因细胞系或重组菌在驱动GUS基因转录方面的应用。
5.如权利要求4所述的应用,其特征在于,所述棉花启动子GbU6-7PS驱动GUS基因在棉花花粉中的高效转录。
6.一种GUS融合表达载体,其特征在于,该表达载体是通过将所述棉花启动子GbU6-7PS与GUS基因连接后,克隆至植物表达载体制备得到。
7.如权利要求6所述的GUS融合表达载体,其特征在于,所述植物表达载体为pCAMBIA1300。
8.权利要求6或7所述的GUS融合表达载体在驱动GUS基因转录方面的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510885400.XA CN105779448B (zh) | 2015-12-04 | 2015-12-04 | 一种棉花启动子GbU6-7PS及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510885400.XA CN105779448B (zh) | 2015-12-04 | 2015-12-04 | 一种棉花启动子GbU6-7PS及应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105779448A true CN105779448A (zh) | 2016-07-20 |
CN105779448B CN105779448B (zh) | 2018-11-27 |
Family
ID=56390185
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510885400.XA Expired - Fee Related CN105779448B (zh) | 2015-12-04 | 2015-12-04 | 一种棉花启动子GbU6-7PS及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105779448B (zh) |
Cited By (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
CN108203714A (zh) * | 2016-12-20 | 2018-06-26 | 华中农业大学 | 一种棉花基因的编辑方法 |
US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
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 |
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 |
US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains 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 |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
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 |
-
2015
- 2015-12-04 CN CN201510885400.XA patent/CN105779448B/zh not_active Expired - Fee Related
Non-Patent Citations (3)
Title |
---|
ARIEL ERIJMAN等: "Transfer-PCR (TPCR): A highway for DNA cloning and protein engineering", 《JOURNAL OF STRUCTURAL BIOLOGY》 * |
SVEIN VALLA等: "《DNA Cloning and Assembly Methods》", 20 January 2014, HUMANA PRESS * |
雷建峰等: "棉花花粉中高效转录U6启动子的克隆及功能分析", 《中国农业科学》 * |
Cited By (43)
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 |
US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
US10954548B2 (en) | 2013-08-09 | 2021-03-23 | 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 |
US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
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 |
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 |
US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
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 |
US10113163B2 (en) | 2016-08-03 | 2018-10-30 | 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 |
CN108203714A (zh) * | 2016-12-20 | 2018-06-26 | 华中农业大学 | 一种棉花基因的编辑方法 |
CN108203714B (zh) * | 2016-12-20 | 2021-03-02 | 华中农业大学 | 一种棉花基因的编辑方法 |
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) |
US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | 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 |
US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11795452B2 (en) | 2019-03-19 | 2023-10-24 | 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 |
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 |
Also Published As
Publication number | Publication date |
---|---|
CN105779448B (zh) | 2018-11-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN105779448B (zh) | 一种棉花启动子GbU6-7PS及应用 | |
CN105779449B (zh) | 一种棉花启动子GbU6-5PS及应用 | |
Jin et al. | Construction of a shuttle vector using an endogenous plasmid from the cyanobacterium Synechocystis sp. PCC6803 | |
Khan et al. | Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus | |
CN106854239B (zh) | 小麦条锈病抗性相关蛋白TaSPX-MFS及其编码基因与应用 | |
CN104946648A (zh) | 一种植物花粉特异性启动子pchf32及其应用 | |
AU2022342157A1 (en) | Class ii, type v crispr systems | |
Reed et al. | Shotgun sequencing of the negative-sense RNA genome of the rhabdovirus Maize mosaic virus | |
CN101413006B (zh) | 一种干旱诱导的水稻花特异性启动子及其应用 | |
CN104630228B (zh) | 棉花热激转录因子基因GhHsf39的启动子及其应用 | |
CN105823888B (zh) | 一种利用甘蔗条纹花叶病毒p3n-pipo构建的亚细胞定位试剂盒 | |
CN107365772B (zh) | 一种植物花粉特异性启动子psp1及其应用 | |
CN109762815A (zh) | 一种在水稻花药与花粉中特异表达的启动子pchf17及其应用 | |
JP5804601B2 (ja) | 藻類を形質転換するために用いられる新規プロモーター | |
WO2023028348A1 (en) | Enzymes with ruvc domains | |
CN104152454B (zh) | 来源于大豆的干旱诱导启动子GmMYB363P及其应用 | |
CN104513830B (zh) | 一种适用于氧化葡萄糖酸杆菌的基因表达载体及其应用 | |
CN110055252B (zh) | 一种在水稻花粉中特异表达的启动子pchf45及其应用 | |
Lehmeyer et al. | A cis-regulatory sequence from a short intergenic region gives rise to a strong microbe-associated molecular pattern-responsive synthetic promoter | |
CN110643608B (zh) | 一种具有花药组织特异性的启动子 | |
CN106244623B (zh) | 可诱导剔除选择标记基因的pBLCK载体及其构建方法 | |
CN105823890B (zh) | 一种利用高粱花叶病毒p3n‑pipo构建的亚细胞定位试剂盒 | |
CN107012143B (zh) | 一种启动子及其应用 | |
CN117737065A (zh) | 一种水曲柳强启动子及其应用 | |
CN105823889B (zh) | 一种利用甘蔗花叶病毒p3n-pipo构建的亚细胞定位试剂盒 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20181127 Termination date: 20191204 |