CN106755097A - 一种山羊tlr4基因敲除载体及其构建方法 - Google Patents
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Abstract
本发明公开了一种山羊TLR4基因敲除载体及其构建方法，采用CRISPR/cas9系统，首先设计TLR4基因的sgRNA片段,合成sgRNA核苷酸序列，构建同时表达sgRNA和Cas9 D10A的质粒PYSY‑sgRNA，连接并转化至大肠杆菌DH5α感受态细胞，最后对转化子进行验证；酶切和测序鉴定证明TLR4基因敲除载体构建正确。本发明采用CRISPR/Cas9构建的载体，为后续获得山羊TLR4基因缺失型肺泡上皮细胞系，研究支原体肺炎感染的免疫应答分子机制提供理论依据。
Description
技术领域
本发明属于基因工程技术领域，尤其涉及一种山羊TLR4基因敲除载体及其构建方法。
背景技术
Toll样受体(TLRs)是天然免疫系统中的一种重要模式识别受体，可选择性识别病原微生物而启动天然免疫,在宿主天然免疫和获得性免疫中具有重要作用。TLR4是TLRs家族的重要成员之一，是内毒素脂多糖(LPS)最重要的模式识别受体，在介导阴性菌及其LPS的宿主反应中发挥关键作用。
CRISPR/Cas9系统是细菌在噬菌体长期的选择压力下进化出来的一种有效抵御外源DNA入侵的免疫机制之一。在菌体内，CRISPR簇在其前导区的调控下转录成precrRNA，并在tracrRNA和Cas9参与下加工成成熟的crRNA，引导crRNA/tracrRNA/Cas9复合体识别结合外源DNA特定序列，剪切DNA双链，从而沉默外源基因的表达。CRISPR/Cas9系统被开发成了一种新型的基因打靶系统。相对于较早的RNAi、ZFN和TALEN系统，这种新型打靶系统具有操作简单、成本低、效率高、可同时沉默任意数量基因等优点。目前，该项技术已经应用于细菌、斑马鱼、小鼠、大鼠、家蚕以及哺乳动物和人类细胞系等，且都表现出较强的基因组编辑活性。Hu等(2014)应用该技术对山羊成纤维细胞中NUP155基因进行了敲除，发现23个单细胞克隆中有5个发生了NUP155基因突变。Wang等(2015)采用CRISPR/Cas9技术对山羊MSTN和FGF5基因进行了敲除。以上研究提示该技术可以成功用于山羊基因敲除。
目前采用ZFNs或TALENs进行基因敲除，对每个基因位点编辑都需要设计和组装两个核酸酶,构建技术难度较大、构建组装时间较长。另外，传统基因敲除，主要是应用基因重组原理通过插入突变和靶向技术使目的基因功能丧失。
然而，目前在山羊上关于TLR4基因敲除及相关功能研究尚未见报道。
发明内容
本发明的目的在于提供一种山羊TLR4基因敲除载体及其构建方法，旨在解决目前采用ZFNs或TALENs进行基因敲除，对每个基因位点编辑都需要设计和组装两个核酸酶,因而构建技术难度较大、构建组装时间较长；而且传统基因敲除，主要是应用基因重组原理通过插入突变和靶向技术使目的基因功能丧失的问题。
本发明是这样实现的，一种山羊TLR4基因敲除载体，该山羊TLR4基因敲除载体的sgRNA核苷酸序列为：
TLR4-gRNA-Lg1：GACTCATATTCAGCACCTGAAGG；
TLR4-gRNA-Rg1：TCACAACAAACTCTTGTCATTGG。
一种引物Oligo，该引物Oligo序列为：
F-TLR4-gRNA-L1：CACCGACTCATATTCAGCACCTGA；
R-TLR4-gRNA-L1：AAACTCAGGTGCTGAATATGAGTC；
F-TLR4-gRNA-R1：CACCGTCACAACAAACTCTTGTCAT；
R-TLR4-gRNA-R1：AAACATGACAAGAGTTTGTTGTGAC。
一种表达sgRNA核苷酸的质粒PYSY-sgRNA，该质粒PYSY-sgRNA为：pYSY-CMV-Cas9n-U6-TLR4-gRNA-L2-SV40-Neo质粒和pYSY-CMV-Cas9n-U6-TLR4-gRNA-R2-EF1a-eGFP质粒。
本发明另一目的在于提供一种山羊TLR4基因敲除载体的构建方法，该山羊TLR4基因敲除载体的构建方法包括：
采用CRISPR/cas9系统，首先设计TLR4基因的sgRNA片段,合成sgRNA核苷酸序列；
构建同时表达sgRNA和Cas9D10A的质粒PYSY-sgRNA，连接并转化至大肠杆菌DH5α感受态细胞；
最后对转化子进行验证。
进一步，该山羊TLR4基因敲除载体的构建方法具体包括：
引物退火：将1ul 100uM的F-Oligo、1ul的100uM R-Oligo、8ul YSY oligo退火缓冲液混合于PCR管内，在PCR仪中以每分钟1.5℃逐渐从95℃降至22℃；
连接：0.5ul退火产物，1ul YSY线性化三合一CRISPR/Cas9n质粒，1ul T4连接酶，2ul 5*T4Buffer，5.5ul Milli Q；
转化：室温15min后用pfu≥108的大肠杆菌DH5a感受态细胞进行转化；
转化子验证：转化涂板后挑取单克隆进行10ul体系菌液PCR验证；
经PCR初步鉴定后进行测序；
菌液37℃过夜培养，无内毒素质粒大提试剂盒提取质粒，并采用微量紫外分光光度计测定质粒浓度。
进一步，所述转化涂板后挑取单克隆进行10ul体系菌液PCR验证，具体包括：挑取单克隆0.5ul菌液，0.5ul YSY验证正向引物，0.5ul R-Oligo，5ulMastermix，3.5ulMilliQ；PCR反应条件为：1)：95℃预变性2mim；2)：94℃变性30s；3)：56℃退火30s；4)：72℃延伸30s；步骤2)到步骤4)运行35个循环；5)：72℃再延伸10min；6)：4℃保存。
本发明提供的山羊TLR4基因敲除载体及其构建方法中CRISPR/Cas9系统，是新开发的一种新型的基因打靶系统，利用细菌或古生菌中存在的的CRISPR簇，在其前导区的调控下转录成precrRNA，并在tracrRNA和Cas9参与下加工成成熟的crRNA，cRNA和tracrRNA二者结合形成的复合物成为，sgRNA与Cas9核酸内切酶结合,并引导其识别结合外源DNA特定序列，剪切DNA双链，从而沉默外源基因的表达。
本发明采用CRISPR/Cas9系统构建TLR4基因敲除载体，方法简单快捷，只需针对该基因敲除位点设计一个长约20bp左右的sgRNA，然后连接通用的Cas9基因即可，而采用ZFNs或TALENs进行基因敲除，对每个基因位点编辑都需要设计和组装两个核酸酶,构建技术难度较大、构建组装时间较长。因此，与传统的ZFNs、TALENs等基因敲除技术比较而言，采用CRISPR/Cas9构建基因敲除载体更为简单快捷，便于进一步推广和应用于后续实验。
另外，传统基因敲除，主要是应用基因重组原理通过插入突变和靶向技术使目的基因功能丧失，与ZFN和TALEN这两种人工核酸酶相比，CRISPR/Cas9系统中的Cas9作为切口酶，具有单链切割活性，可以在特定位置制造单链切口，这样基本不会引起非同源末端连接，从而高效地介导外源基因的定点敲入，或对基因组进行点突变，大大降低了非同源末端连接所带来的风险。
本发明构建的载体，利用RNA导向的CRISPR-Cas9系统形成双切口，在未影响靶向切割效率的前提下大大降低了脱靶效应，提高基因敲除效率。
本发明采用CRISPR/Cas9系统构建TLR4敲除载体，为后续获得山羊TLR4基因缺失型肺泡上皮细胞系、研究山羊肺炎支原体感染的免疫应答分子机制提供理论依据。
附图说明
图1是本发明实施例提供的山羊TLR4基因敲除载体的构建方法流程图；
图2是本发明实施例提供的PCR验证克隆电泳图片示例一；
图中：Marker：Trans 2K Plus DNA Marker，从下到上依次为100bp,250bp,500bo,750bp,1000bp,3000bp,5000bp；2:阴性对照；3-6:TLR4-gRNA-Rg1克隆。
图3是本发明实施例提供的PCR验证克隆电泳图片示例二；
图中：Marker：Trans 2K Plus DNA Marker，从下到上依次为100bp,250bp,500bo,750bp,1000bp,3000bp,5000bp；2:阴性对照；3-6:TLR4-gRNA-Lg1克隆。
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。
下面结合附图对本发明的应用原理作进一步描述。
本发明实施例提供的山羊TLR4基因敲除载体，该山羊TLR4基因敲除载体的sgRNA核苷酸序列为：
SEQ ID NO1：TLR4-gRNA-Lg1：GACTCATATTCAGCACCTGAAGG；
SEQ ID NO2：TLR4-gRNA-Rg1：TCACAACAAACTCTTGTCATTGG。
一种引物Oligo，该引物Oligo序列为：
SEQ ID NO3：F-TLR4-gRNA-L1：CACCGACTCATATTCAGCACCTGA；
SEQ ID NO4：R-TLR4-gRNA-L1：AAACTCAGGTGCTGAATATGAGTC；
SEQ ID NO5：F-TLR4-gRNA-R1：CACCGTCACAACAAACTCTTGTCAT；
SEQ ID NO6：R-TLR4-gRNA-R1：AAACATGACAAGAGTTTGTTGTGAC。
本发明提供的一种表达sgRNA核苷酸的质粒PYSY-sgRNA，该质粒PYSY-sgRNA为：pYSY-CMV-Cas9n-U6-TLR4-gRNA-L2-SV40-Neo质粒和pYSY-CMV-Cas9n-U6-TLR4-gRNA-R2-EF1a-eGFP质粒。
如图1所示：本发明实施例提供的山羊TLR4基因敲除载体的构建方法，该山羊TLR4基因敲除载体的构建方法包括：
S101：采用CRISPR/cas9系统，首先设计TLR4基因的sgRNA片段,合成sgRNA核苷酸序列；
S102：构建同时表达sgRNA和Cas9D10A的质粒PYSY-sgRNA，连接并转化至大肠杆菌DH5α感受态细胞；
S103：最后对转化子进行验证。
进一步，该山羊TLR4基因敲除载体的构建方法具体包括：
引物退火：将1ul 100uM的F-Oligo、1ul的100uM R-Oligo、8ul YSY oligo退火缓冲液混合于PCR管内，在PCR仪中以每分钟1.5℃逐渐从95℃降至22℃；
连接：0.5ul退火产物，1ul YSY线性化三合一CRISPR/Cas9n质粒，1ul T4连接酶，2ul 5*T4Buffer，5.5ul Milli Q；
转化：室温15min后用pfu≥108的大肠杆菌DH5a感受态细胞进行转化；
转化子验证：转化涂板后挑取单克隆进行10ul体系菌液PCR验证：0.5ul菌液，0.5ul YSY验证正向引物，0.5ul R-Oligo，5ul Mastermix，3.5ul MilliQ；
PCR反应条件为：
1)：95℃预变性2mim；
2)：94℃变性30s；
3)：56℃退火30s；
4)：72℃延伸30s；步骤2)到步骤4)运行35个循环；
5)：72℃再延伸10min；
6)：4℃保存；
经PCR初步鉴定后进行测序；
菌液37℃过夜培养，无内毒素质粒大提试剂盒提取质粒，并采用微量紫外分光光度计测定质粒浓度。
下面结合试验方法对本发明的应用原理作进一步描述
1材料和方法
1.1材料
Trans2K Plus DNA Marker购自全式金生物技术有限公司，CRISPR/Cas9n质粒购自南京尧舜禹公司，大肠杆菌DH5a感受态细胞、T4连接酶、无内毒素质粒大提试剂盒均购自天根生化有限公司。阳离子脂质体2000(Invitrogen)。PCR引物由上海铂尚生物技术有限公司合成。
1.2方法
1.2.1sgRNA靶点确定和引物设计
根据山羊TLR4基因(GenBank:HQ263215.1)，找到CDS序列并分析其结构，根据该基因结构确定敲除位点，选择外显子序列输入到软件中，得到gRNA序列。并根据设计生成的sgRNA，设计并合成对应的引物Oligo如下：
F-TLR4-gRNA-L1：CACCGACTCATATTCAGCACCTGA；
R-TLR4-gRNA-L1：AAACTCAGGTGCTGAATATGAGTC；
F-TLR4-gRNA-R1：CACCGTCACAACAAACTCTTGTCAT；
R-TLR4-gRNA-R1：AAACATGACAAGAGTTTGTTGTGAC；
1.2.2CRISPR/Cas9基因敲除载体的构建
引物退火：1ul F-Oligo(100uM)，1ul R-Oligo(100uM)，8ul YSY oligo退火缓冲液，以上溶液混合于PCR管内，在PCR仪中以每分钟1.5℃逐渐从95℃降至22℃。
连接：0.5ul退火产物，1ul YSY线性化三合一CRISPR/Cas9n质粒，1ul T4连接酶，2ul 5*T4Buffer，5.5ul Milli Q。
转化：室温15min后用大肠杆菌DH5a感受态细胞(pfu≥108)进行转化。
转化子验证：转化涂板后挑取单克隆进行10ul体系菌液PCR验证。
0.5ul菌液，0.5ul YSY验证正向引物，0.5ul R-Oligo，5ul Mastermix，3.5ulMilliQ；
PCR反应条件：Seg1：95℃预变性2mim；Seg2：94℃变性30s；Seg3：56℃退火30s；Seg4：72℃延伸30s；Seg2to Seg4运行35个循环；Seg5：72℃再延伸10min；Seg6：4℃保存。
经PCR初步鉴定为阳性克隆送至南京金斯瑞生物科技有限公司进行测序。
菌液37℃过夜培养，无内毒素质粒大提试剂盒提取质粒，并采用微量紫外分光光度计测定质粒浓度。
2.结果
2.1sgRNA设计
在U6启动子驱动下设计的sgRNA转录本RNA序列分别为：
TLR4-gRNA-Lg1：GACTCATATTCAGCACCTGAAGG；
TLR4-gRNA-Rg1：TCACAACAAACTCTTGTCATTGG；
2.2PCR验证阳性克隆电泳图片如图2、3所示。
2.3送检测序结果
将之前验证正确的阳性克隆菌液送至金斯瑞生物技术有限公司测序，测序部分序列比对结果具体如下：
2.3.1pYSY-CMV-Cas9n-U6-TLR4-gRNA-L2-SV40-Neo；
通过对比，其同源性达到100％。
2.3.2pYSY-CMV-Cas9n-U6-TLR4-gRNA-R2-EF1a-Egfp；
其同源性达到100％。因此，通过以上序列比对，可确定目标质粒构建成功。
2.4山羊TLR4MC1R基因的敲除质粒对提取及浓度测定：
pYSY-CMV-Cas9n-U6-TLR4-gRNA-L2-SV40-Neo质粒：4ug浓度:130ng/ul；
pYSY-CMV-Cas9n-U6-TLR4-gRNA-R2-EF1a-eGFP质粒：4ug浓度:209ng/ul。
下面结合原理分析对本发明进一步说明。
CRISPR/Cas9系统，是新开发的一种新型的基因打靶系统，利用细菌或古生菌中存在的的CRISPR簇，在其前导区的调控下转录成precrRNA，并在tracrRNA和Cas9参与下加工成成熟的crRNA，cRNA和tracrRNA二者结合形成的复合物成为，sgRNA与Cas9核酸内切酶结合,并引导其识别结合外源DNA特定序列，剪切DNA双链，从而沉默外源基因的表达。
本发明采用CRISPR/Cas9系统构建TLR4基因敲除载体，方法简单快捷，只需针对该基因敲除位点设计一个长约20bp左右的sgRNA，然后连接通用的Cas9基因即可，而采用ZFNs或TALENs进行基因敲除，对每个基因位点编辑都需要设计和组装两个核酸酶,构建技术难度较大、构建组装时间较长。因此，与传统的ZFNs、TALENs等基因敲除技术比较而言，采用CRISPR/Cas9构建基因敲除载体更为简单快捷，便于进一步推广和应用于后续实验。
另外，传统基因敲除，主要是应用基因重组原理通过插入突变和靶向技术使目的基因功能丧失，与ZFN和TALEN这两种人工核酸酶相比，CRISPR/Cas9系统中的Cas9作为切口酶，具有单链切割活性，可以在特定位置制造单链切口，这样基本不会引起非同源末端连接，从而高效地介导外源基因的定点敲入，或对基因组进行点突变，大大降低了非同源末端连接所带来的风险。
本发明构建的载体，利用RNA导向的CRISPR-Cas9系统形成双切口，在未影响靶向切割效率的前提下大大降低了脱靶效应，提高基因敲除效率。
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。
<110> 安徽省农业科学院畜牧兽医研究所
<120> 一种山羊TLR4基因敲除载体的构建方法
<160> 6
<210> 1
<211>23
<212> RNA
<213>人工序列
<400> 核苷酸序列
GACTCATATTCAGCACCTGAAGG
<210> 2
<211>23
<212> RNA
<213>人工序列
<400>核苷酸序列
TCACAACAAACTCTTGTCATTGG
<210> 3
<211>24
<212> RNA
<213>人工序列
<400>核苷酸序列
CACCGACTCATATTCAGCACCTGA
<210> 4
<211>24
<212> RNA
<213>人工序列
<400>核苷酸序列
AAACTCAGGTGCTGAATATGAGTC
<210> 5
<211>24
<212> RNA
<213>人工序列
<400>核苷酸序列
CACCGTCACAACAAACTCTTGTCAT
<210> 6
<211>24
<212> RNA
<213>人工序列
<400>核苷酸序列
AAACATGACAAGAGTTTGTTGTGAC
Claims (6)
1.一种山羊TLR4基因敲除载体，其特征在于，该山羊TLR4基因敲除载体的sgRNA核苷酸序列为：SEQ ID NO1和SEQ ID NO2。
2.一种包含权利要求1所述的sgRNA核苷酸合成对应的引物Oligo，其特征在于，该引物Oligo序列为：SEQ ID NO3、SEQ ID NO4、SEQ ID NO5、SEQ ID NO6。
3.一种表达权利要求1所述的sgRNA核苷酸的质粒PYSY-sgRNA，其特征在于，该质粒PYSY-sgRNA为：
pYSY-CMV-Cas9n-U6-TLR4-gRNA-L2-SV40-Neo质粒和
pYSY-CMV-Cas9n-U6-TLR4-gRNA-R2-EF1a-eGFP质粒。
4.一种如权利要求1所述的山羊TLR4基因敲除载体的构建方法，其特征在于，该山羊TLR4基因敲除载体的构建方法包括：
采用CRISPR/cas9系统，首先设计TLR4基因的sgRNA片段,合成sgRNA核苷酸序列；
构建同时表达sgRNA和Cas9D10A的质粒PYSY-sgRNA，连接并转化至大肠杆菌DH5α感受态细胞；
最后对转化子进行验证。
5.如权利要求4所述的山羊TLR4基因敲除载体的构建方法，其特征在于，该山羊TLR4基因敲除载体的构建方法具体包括：
引物退火：将1ul 100uM的F-Oligo、1ul的100uM R-Oligo、8ul YSY oligo退火缓冲液混合于PCR管内，在PCR仪中以每分钟1.5℃逐渐从95℃降至22℃；
连接：0.5ul退火产物，1ul YSY线性化三合一CRISPR/Cas9n质粒，1ul T4连接酶，2ul5*T4Buffer，5.5ul Milli Q；
转化：室温15min后用pfu≥108的大肠杆菌DH5a感受态细胞进行转化；
转化子验证：转化涂板后挑取单克隆进行10ul体系菌液PCR验证；
经PCR初步鉴定后进行测序；
菌液37℃过夜培养，无内毒素质粒大提试剂盒提取质粒，并采用微量紫外分光光度计测定质粒浓度。
6.如权利要求5所述的山羊TLR4基因敲除载体的构建方法，其特征在于，所述转化涂板后挑取单克隆进行10ul体系菌液PCR验证，具体包括：挑取单克隆0.5ul菌液，0.5ul YSY验证正向引物，0.5ul R-Oligo，5ul Mastermix，3.5ul MilliQ；PCR反应条件为：1)：95℃预变性2mim；2)：94℃变性30s；3)：56℃退火30s；4)：72℃延伸30s；步骤2)到步骤4)运行35个循环；5)：72℃再延伸10min；6)：4℃保存。
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Cited By (33)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| 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 |
| CN109651498A (zh) * | 2018-12-07 | 2019-04-19 | 上海浩港生物技术有限公司 | 一种lbp相关蛋白质多肽抗原的制备方法 |
| 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 |
| CN111321171A (zh) * | 2018-12-14 | 2020-06-23 | 江苏集萃药康生物科技有限公司 | 一种应用CRISPR/Cas9介导ES打靶技术制备基因打靶动物模型的方法 |
| 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 |
| 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 |
| US12157760B2 (en) | 2018-05-23 | 2024-12-03 | The Broad Institute, Inc. | Base editors and uses thereof |
| US12281338B2 (en) | 2018-10-29 | 2025-04-22 | The Broad Institute, Inc. | Nucleobase editors comprising GeoCas9 and uses thereof |
| US12351837B2 (en) | 2019-01-23 | 2025-07-08 | The Broad Institute, Inc. | Supernegatively charged proteins and uses thereof |
| US12390514B2 (en) | 2017-03-09 | 2025-08-19 | President And Fellows Of Harvard College | Cancer vaccine |
| US12406749B2 (en) | 2017-12-15 | 2025-09-02 | The Broad Institute, Inc. | Systems and methods for predicting repair outcomes in genetic engineering |
| US12435330B2 (en) | 2019-10-10 | 2025-10-07 | The Broad Institute, Inc. | Methods and compositions for prime editing RNA |
| US12473543B2 (en) | 2019-04-17 | 2025-11-18 | The Broad Institute, Inc. | Adenine base editors with reduced off-target effects |
| US12509680B2 (en) | 2023-05-31 | 2025-12-30 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820454A (zh) * | 2014-03-04 | 2014-05-28 | 黄行许 | CRISPR-Cas9特异性敲除人PD1基因的方法以及用于特异性靶向PD1基因的sgRNA |
| CN106715431A (zh) * | 2014-09-16 | 2017-05-24 | 吉利德科学公司 | Toll样受体调节剂的固体形式 |
-
2016
- 2016-12-27 CN CN201611222780.XA patent/CN106755097A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820454A (zh) * | 2014-03-04 | 2014-05-28 | 黄行许 | CRISPR-Cas9特异性敲除人PD1基因的方法以及用于特异性靶向PD1基因的sgRNA |
| CN106715431A (zh) * | 2014-09-16 | 2017-05-24 | 吉利德科学公司 | Toll样受体调节剂的固体形式 |
Non-Patent Citations (6)
| Title |
|---|
| FERGUSON C等: "Toll-like receptor 4 (Tlr4) knockout rats produced by transcriptional activator-like effectornuclease (TALEN)-mediated gene inactivation", 《ALCOHOL》 * |
| RAJA A等: "Sequence analysis of Toll-like receptor genes 1-10 of goat (Capra hircus)", 《VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY》 * |
| 刘健: "CRISPR-Cas9技术介导阿尔巴斯白绒山羊MSTN基因敲除的研究", 《中国优秀硕士学位论文全文数据库》 * |
| 徐燕: "山羊Toll样受体基因家族的克隆表达及群体进化分析研究", 《中国优秀硕士学位论文全文数据库》 * |
| 王正国等: "《分子创伤学》", 31 August 2004, 福建科学技术出版社 * |
| 王雪芹: "《运动诱导大鼠免疫细胞凋亡及机制研究》", 31 March 2016, 山东人民出版社 * |
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| US12509680B2 (en) | 2023-05-31 | 2025-12-30 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
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