CN113774077A - 一种应用于结核分枝杆菌的单碱基基因编辑***及方法 - Google Patents
一种应用于结核分枝杆菌的单碱基基因编辑***及方法 Download PDFInfo
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Abstract
本发明公开了一种应用于结核分枝杆菌的单碱基基因编辑***及方法,利用CRISPR技术与胞嘧啶核苷脱氨酶***偶联,实现了对结核分枝杆菌基因的高效的遗传操作。本发明的单碱基基因编辑***通过抑制RecA介导的同源重组和NucS介导的错配修复来提升碱基编辑的效率,能够实现G:C到A:T的单个碱基替换以及使目标基因翻译提前终止,为研究结核分枝杆菌提供了有效的技术手段。
Description
技术领域
本发明涉及基因编辑***,特别涉及一种基于CRISPR/Cas***的胞嘧啶核苷脱氨技术对结核分枝杆菌进行单碱基基因编辑的***和方法。
背景技术
结核分枝杆菌(mycobacterium tuberculosis)是结核病(TB)的病原体,是单一病原体导致死亡的主要原因。世界卫生组织(WHO)估计,2019年,全球有1000万例新发结核病患者。耐多药结核(Multidrug-resistant TB,MDR-TB)和广泛耐药结核(Extensivelydrug-resistant TB,XDR-TB)菌株的出现是一个持续存在的健康问题,迫切需要新的治疗策略。药物靶标的识别和表征强烈依赖于有效的基因操作技术。然而,分枝杆菌的遗传操作极具挑战性——主要是因为它们缓慢的生长速度、致病性,以及高GC含量的基因组。因此,当前急需一种高效且实用的方法对结核分枝杆菌进行基因编辑,了解其相关耐药机制,并对临床治疗做出指导。
近年来,分枝杆菌基因编辑技术快速发展,特别是得益于CRISPR-Cas***的出现。一种基于同源重组(homologous recombination,HR)和CRISPR-Cas12a介导的基因编辑***可以在耻垢分枝杆菌中快速产生点突变、缺失和***。此外,一种CRISPR-Cas介导的非同源末端连接(non-homologous end joining,NHEJ)基因组编辑工具,允许在耻垢分枝杆菌和结核分枝杆菌基因组上产生无标记的碱基缺失。然而,这两种方法都不能在结核分枝杆菌中用于引入点突变或进行精确的基因操作。
单碱基编辑器的发现为精确的基因操作提供了一种新的策略。该方法不同于HR和NHEJ需要引入DNA双链断裂(double-strand breaks,DSBs)或供体DNA模板。目前的碱基编辑器主要包含催化活性受损的Cas核酸酶和单链DNA脱氨酶——胞嘧啶核苷脱氨酶(如APOBEC)或腺嘌呤核苷脱氨酶(TadA)。胞嘧啶核苷脱氨酶特异性催化胞嘧啶(C)转化为尿嘧啶(U),尿嘧啶被DNA聚合酶读取为胸腺嘧啶(T);腺嘌呤核苷脱氨酶将腺嘌呤(A)转化为肌苷(I),后者被读作鸟嘌呤(G)。在胞嘧啶碱基编辑器(cytosine base editors,CBEs)中,将APOBEC与催化活性受损的Cas9蛋白相融合,在具有靶向作用的sgRNA引导下到达目标基因位置时,可以形成由蛋白质-RNA-DNA构成的“R-loop”结构,暴露的单链DNA窗口可作为APOBEC发挥作用的底物,窗口内任何胞嘧啶核苷均可发生脱氨基作用,并形成U:G中间体。在细菌细胞中,DNA形成U:G中间体时,通常由尿嘧啶N-糖基化酶(uracil DNAglycosylase,UNG)发挥碱基切除修复(base excision repair,BER)作用来切除碱基U,为了保护编辑后的U:G中间体免于被UNG切除,可以将尿嘧啶DNA糖基化酶抑制子(uracil DNAglycosylase inhibitor,UGI)直接与催化活性受损的Cas9的C末端融合在一起,其可以抑制UNG发挥碱基切除修复,并促进细胞发生错配修复形成U:A中间体,进而在DNA复制时识别为T:A,从而实现了目标位点碱基C到T的转换。此外,通过催化特殊密码子(CAA、CAG、CGA或TGG)的转换,可以通过产生一个提前终止的密码子来破坏目的基因,从而失活基因功能。为了提高碱基编辑效率,大多数碱基编辑器将无核酸酶活性的Cas9(dead Cas9,dCas9)替换为Cas9 nickase(nCas9)。
胞嘧啶碱基编辑***已经在大肠杆菌、金黄色葡萄球菌、肺炎克雷伯菌、链霉菌等微生物中发展和应用,但在分枝杆菌中还没有开发。如果能够实现对结核分枝杆菌单碱基基因编辑,该技术将与CRISPR-HR和CRISPR-NHEJ相互补充,共同实现结核分枝杆菌基因组各种遗传操作。
发明内容
本发明旨在通过抑制同源重组和错配修复两种DNA修复途径,利用CRISPR-Cas***与胞嘧啶核苷脱氨酶APOBEC1***相偶联,实现对结核分枝杆菌单碱基基因编辑。
本发明包括以下两个方面:1)抑制RecA介导的同源重组和NucS介导的错配修复以提高单碱基编辑效率。由于nCas9sth仍然保留着nickase活性,它可以切断DNA的非编辑链,而单链断裂可以启动同源重组DNA修复途径,因此通过高表达RecA抑制蛋白RecX抑制该途径可以提高单碱基编辑效率;DNA错配修复可以将脱氨基后的碱基切除,而结核分枝杆菌NucS最近被发现具有典型错配修复的特征,因此利用NucS显性复性突变体抑制该途径可以提高单碱基编辑效率;2)诱导表达单碱基编辑元件nCas9sth-APOBEC1-UGI对目标基因进行单碱基编辑。
在本发明的第一方面,提供了一种应用于结核分枝杆菌的单碱基基因编辑***,是一种双质粒的CRISPR-Cas辅助的胞嘧啶核苷脱氨编辑***,包括单碱基编辑器质粒和抑制DNA修复元件质粒,其中,所述单碱基编辑器质粒包含sgRNA作用元件和编码nCas9sth、胞嘧啶核苷脱氨酶及尿嘧啶DNA糖基化酶抑制子形成的融合蛋白的基因,所述抑制DNA修复元件质粒包含降低RecA依赖的同源重组的编码RecX的基因元件和降低NucS依赖的错配修复的编码NucSE107A的基因元件。
所述单碱基编辑器质粒是复制性穿梭载体,在本发明的一个实施例中,所述复制性穿梭载体含有分枝杆菌复制起点oriM、大肠杆菌复制起点oriE和sacB反向筛选标记。
所述单碱基编辑器质粒上编码nCas9sth的基因为来源于嗜热链球菌(Streptococcus thermophilus)的cas9sth1基因,其蛋白质序列的第九位天冬氨酸突变为丙氨酸。
所述胞嘧啶核苷脱氨酶基因是APOBEC1基因,本发明实施例中应用的优化后的基因序列如序列表中SEQ ID No:9所示。
所述尿嘧啶DNA糖基化酶抑制子基因是UGI基因,本发明实施例中应用的优化后的基因序列如序列表中SEQ ID No:10所示。
在本发明的一个实施例中,所述单碱基编辑器质粒上编码的融合蛋白为APOBEC1通过一个长为16个氨基酸(Ser-Gly-Ser-Glu-Thr-Pro-Gly-Thr-Ser-Glu-Ser-Ala-Thr-Pro-Glu-Ser)(SEQ ID No:17)的连接子连接到nCas9sth的N端;UGI通过一个长为4个氨基酸(Ser-Gly-Gly-Ser)(SEQ ID No:18)的连接子连接到nCas9sth的C端。
所述单碱基编辑器质粒上的融合蛋白基因由诱导型启动子(例如PtetO)驱动表达。
所述抑制DNA修复元件质粒表达的NucSE107A为NucS蛋白序列第107位谷氨酸突变为丙氨酸的突变体。
所述抑制DNA修复元件质粒上的编码RecX的基因和编码NucSE107A的基因由诱导型启动子(例如PtetO)驱动表达。
在本发明的另一方面,基于上述单碱基基因编辑***提供了一种对结核分枝杆菌进行单碱基基因编辑的方法,包括以下步骤:
1)用所述抑制DNA修复元件质粒转化结核分枝杆菌,并将转化子制备成感受态细胞;
2)将靶向目标基因片段的sgRNA序列连接到所述单碱基编辑器质粒上的sgRNA作用元件中,得到含有sgRNA序列的单碱基编辑器质粒;
3)用步骤2)制备的含有sgRNA序列的单碱基编辑器质粒转化步骤1)制备的感受态细胞,诱导相关元件表达进行基因编辑,对得到的转化子进行验证,得到目标基因片段被编辑的转化子。
上述步骤2)中,所述sgRNA序列长度优选为20bp,编辑目标位点位于目标基因片段中PAM序列远端第2-16位,优选为第5、6位。
综上所述,本发明利用CRISPR-Cas***与胞嘧啶核苷脱氨酶APOBEC1***联用,实现了对结核分枝杆菌基因的高效的遗传操作。本发明的单碱基基因编辑***通过抑制RecA介导的同源重组和NucS介导的错配修复来提升碱基编辑的效率,能够实现G:C到A:T的单个碱基替换以及使目标基因翻译提前终止,为研究结核分枝杆菌提供了有效的技术手段。
附图说明
图1.本发明所构建的单碱基编辑***的载体结构示意图,其中:A为抑制DNA修复元件质粒,B为单碱基编辑器质粒。
图2.本发明实施例1对结核分枝杆菌毒素基因进行单碱基突变的测序结果,其中:A、B、C分别对应毒素编码基因Rv0582、Rv0627和Rv2530。
图3.本发明实施例2利用本发明单碱基编辑方法对katG突变及耐药性探究的实验结果,其中:A为katG突变株测序结果,B为野生型菌株和突变株对异烟肼耐药性检测结果。
具体实施方式
下面结合附图,通过实施例详细介绍本发明基因编辑***的具体构建过程,并通过实施例进一步阐述本发明基因编辑***的使用方法和实验结果。
一.质粒***的构建
1.抑制DNA修复元件质粒
如图1中A所示,本发明实施例中的抑制DNA修复元件质粒pRecX-NucSE107A为穿梭质粒,表达来自结核分枝杆菌的RecX蛋白和NucS显性复性突变体NucSE107A,共同由诱导型启动子PtetO诱导表达。该质粒的构建过程如下:
首先,通过PCR获取RecX、NucSE107A、Pmyc1tetO和tetR片段。
以结核分枝杆菌H37Ra的基因组为模板,采用如下引物序列PCR获取RecX的基因:
F for recX:5’-gcgagtaccggctgttctgaaggcggtatcgatatgacgg-3’(SEQ ID No:1);
R for recX:5’-aatttaatcgcggcctcgagttagacgcgtcggcgctccc-3’(SEQ ID No:2)。
以公司构建的含有NucS第107位突变(Glu突变为Ala)的载体为模板,采用如下引物序列PCR获取NucSE107A:
F for NucSE107A:5’-attaagaaggagatatacatgtgagtcgggtgcgtctagt-3’(SEQ IDNo:3);
R for NucSE107A:5’-ccgtcatatcgataccgccttcagaacagccggtactcgc-3’(SEQ IDNo:4)。
以pKM444质粒(参见ORBIT:a New Paradigm for Genetic Engineering ofMycobacterial Chromosomes.mBio.2018Dec 11;9(6):e01467-18.)为模板,采用如下引物序列PCR获取Pmyc1tetO:
F for Pmyc1tetO:5’-catccgtggcgcggccgcgggatccaatattggatcgtcggcacc-3’(SEQ ID No:5);
R for Pmyc1tetO:5’-tagacgcacccgactcacatgtatatctccttcttaattaagcatgc-3’(SEQ ID No:6)。
以PLJR962质粒(参见Programmable transcriptional repression inmycobacteria using an orthogonal CRISPR interference platform.NatMicrobiol.2017Feb 6;2:16274.)为模板,采用如下引物序列PCR获取tetR:
F for tetR:5’-tcttcacgaggcagacctcactagtagaaatattggatcgtcggc-3’(SEQ IDNo:7);
R for tetR:5’-ggggtctgacgctcatggaactagtttaagacccactttcacatt-3’(SEQ IDNo:8)。
PCR产物经琼脂糖凝胶电泳鉴定并回收,tetR回收产物通过无缝连接(seamlessassembly)的方式与PYC1640(含有Zeor的穿梭质粒,参见Programmable transcriptionalrepression in mycobacteria using an orthogonal CRISPR interferenceplatform.Nat Microbiol.2017Feb 6;2:16274.)经SpeI酶切的线性载体连接得到PYC2211。
而后将RecX、NucSE107A、Pmyc1tetO的PCR回收产物通过无缝连接(seamlessassembly)的方式与PYC2211经BamHI、XhoI酶切的线性载体连接得到PYC2214,即抑制DNA修复元件质粒。
2.构建单碱基编辑器质粒
如图1中B所示,本实施例中所述的单碱基编辑器质粒pCBE为穿梭质粒,包含由PtetO诱导表达的APOBEC1-nCas9sth-UGI融合蛋白、sgRNA支架序列和sacB反向筛选标记。构建该质粒时,首先对脱氨酶APOBEC1和UGI的基因进行密码子优化,并克隆到含有nCas9sth1和同源sgRNA支架的质粒中。APOBEC1和UGI优化后的编码基因序列分别如序列表中SEQ IDNo:9和SEQ ID No:10所示。APOBEC1(缺少终止密码子)通过16个氨基酸的柔性连接体(其核苷酸序列见序列表中SEQ ID No:11)融合到nCas9sth1(缺少起始和终止密码子)的N端。UGI(缺少起始密码子)通过4个氨基酸的柔性连接体(其核苷酸序列见序列表中SEQ ID No:12)连接到nCas9sth1的C端。sgRNA的构建通过将退火后的靶向目标基因的sgRNA寡核苷酸序列通过Golden Gate assembly的方式***pCBE的BsmBI位点。
二.结核分枝杆菌单碱基基因编辑实验流程
1、构建含抑制DNA修复元件质粒的菌株
将构建好的抑制DNA修复元件质粒转化进结核分枝杆菌H37Ra中,得到含该质粒的菌株。转化方式为电转化,需要预先制备感受态细胞,步骤如下:
(1)冻存的野生型H37Ra在7H10+OADC平板上复苏,约两到三周后长出菌落。
(2)挑取一个单菌落并把该单菌落接种到含液体培养基(7H9+OADC)的小培养皿中静置培养至饱和,37℃,约15天左右。
(3)将饱和菌液吸取适量转接到300-500mL新鲜7H9+OADC培养液中,使菌液初始OD600约0.02左右,在滚瓶中滚动培养,速度约50rpm,培养温度37℃。
(4)大约5天后,菌液OD600达到0.6至0.8时,加入0.1体积的15%的甘氨酸,继续滚动培养18-24小时。
(5)制备感受态细胞:离心菌液(3700g,10min,室温),弃去上清液。用10%的甘油水反复吹打菌体沉淀,使其重悬均匀。再次离心(3700g,10min,室温),弃去上清液。用体积减半的10%的甘油水吹打重悬菌体沉淀,再次离心,再用体积减半的10%的甘油水吹打,如此反复三次。最后用少量10%的甘油水重悬,每200μL分装到EP管中,-80摄氏度保存备用。
将抑制DNA修复元件质粒电转到上述制备的感受态细胞中,电转参数(电阻1000Ω、电容25μF和电压2.5kV)。电转后加入1mL不含抗生素的7H9+OADC,将菌液转移到离心管中并置于滚瓶中滚动培养24小时。取适量菌液涂在含博来霉素的7H10+OADC平板上。37℃培养两到三周后即可长出单菌落。通过菌落PCR和测序技术验证长出的单菌落是否含有目的质粒。
2、构建靶向目标基因的单碱基编辑器质粒
在目标基因的开放阅读框内寻找合适的PAM序列,从PAM的5’上游第三位开始向上游拉20bp的碱基序列,得到sgRNA top链,top链的反向互补链即为sgRNA bottom链,在两条链的5’端加上BbsI酶切位点。即:
sgRNA top链:5’-GGGA-------------------3’
sgRNA bottom链:5’-AAAC-------------------3’
sgRNA的两条链通过退火连接到原始单碱基编辑器质粒上sgRNA位点,即得到靶向目标基因的单碱基编辑器质粒。
3、转化单碱基编辑器质粒对目标基因进行编辑
将步骤1得到的含抑制DNA修复元件质粒的菌株制备成感受态细胞并转化步骤2得到的靶向目标基因的单碱基编辑器质粒。感受态制备步骤和电转步骤同上。转化后复苏的菌液涂在含博来霉素和卡那霉素以及50ng/mL无水四环素(anhydrotetracycline,ATc)的7H10+OADC平板上,37℃培养两到三周后即可长出单菌落。
4、验证突变效率
挑取单菌落,用针对目标基因序列的特异性引物PCR和测序确认基因是否发生单个碱基替换。
5、丢掉抑制DNA修复元件质粒和单碱基编辑器质粒
将单克隆菌落用1mL 7H9+OADC培养液吹匀,取20μL涂在不含抗生素和含2%蔗糖的7H10平板上,培养约三周后长出单菌落。单碱基编辑器质粒上表达有sacB基因,不能在含蔗糖的平板上生长。将单菌落分别划线在含卡那霉素、博来霉素和不含抗生素的7H10平板上,三周后挑只在无抗平板上生长的单克隆。
实施例1.对结核分枝杆菌毒素基因进行单碱基突变
选择结核分枝杆菌毒素-抗毒素(TA)***的三个毒素编码基因Rv0582、Rv0627和Rv2530作为目标基因进行突变。首先将用上述方法构建的抑制DNA修复元件质粒电转进野生型结核分枝杆菌感受态当中,得到包含该质粒的菌株,再将该菌株制备成感受态。如图2所示设计靶向这些基因的sgRNA序列:靶向Rv0582的sgRNA序列为5’-gcaccgaatcggttgcggttt-3’(SEQ ID No:13),PAM为GGAAT,PAM远端第5位C突变引入提前终止的密码子;靶向Rv0627的sgRNA序列为5’-gggctcaacgcctggcgacc-3’(SEQ ID No:14),PAM为GGAAT,PAM远端第6位C突变可以引入提前终止的密码子;靶向Rv2530的sgRNA序列为5’-gtggtggcccacccattcga-3’(SEQ ID No:15),PAM为AGAAC,PAM远端第9、10位突变可以引入提前终止的密码子,从而使相应的基因失活。合成这些靶标序列并退火连接到pCBE质粒上的sgRNA位点,得到靶向相应目标基因的单碱基编辑器质粒。分别将这些质粒电转化到野生型结核分枝杆菌细胞和上述制备的含有抑制DNA修复元件质粒的细胞中,在无抗生素的培养基中富集24小时后,取适量菌液涂板到含卡那霉素、博来霉素和50ng/mL ATc的7H10+OADC的平板上进行单碱基编辑。培养两到三周后,随机挑取8个转化子通过PCR和测序检测是否发生目标突变。在野生型结核分枝杆菌中8个菌落都没有检测到发生碱基编辑,而如图2的结果显示,在含有抑制DNA修复元件质粒的菌株中,Rv0582有6个菌落发生编辑,其中5个在第4、5、10位的胞嘧啶C突变成胸腺嘧啶T;对Rv0627,8个菌落中有1个在第6、9位发生编辑;对Rv2530,有4个菌落发生预期位点和附近位点突变。三个目标基因的编辑效率分别达到75%、12.5%和50%。
实施例2.单碱基编辑技术应用于结核分枝杆菌耐药研究
异烟肼(Isoniazid,INH)是抗结核药物中最重要的一线药物之一,然而目前全球耐异烟肼的结核分枝杆菌菌株呈上升趋势。对INH的抗性通常是由于Rv1908c基因发生非同义点突变,该基因编码过氧化氢酶过氧化物酶KatG,而KatG是INH激活所必需的。为了验证该单碱基编辑***可以用来鉴定耐药基因,如图3中A所示,我们利用上述单碱基编辑方法对katG基因进行突变,将第595位的精氨酸突变为终止密码子,并评估了野生株和突变株的INH敏感性。设计的sgRNA序列为5’-AGATGGCTTCCGAAACTACC-3’(SEQ ID No:16),PAM为GGAAA,PAM远端第11位C变成T可以引入终止密码子TGA。合成该靶标序列并退火连接到pCBE质粒上的sgRNA位点,得到靶向katG的单碱基编辑器质粒并将该质粒转化到含抑制DNA修复元件质粒的细胞中。在无抗生素的培养基中富集24小时后,取适量菌液涂板到含卡那霉素、博来霉素和50ng/mL ATc的7H10+OADC的平板上进行单碱基编辑。培养两到三周后,随机挑取8个转化子通过PCR和测序检测是否发生目标突变。测序分析显示,随机挑选的8个转化子中有一个在预期的位点(第11位)发生突变,第10位和16位的胞嘧啶也发生了突变。与之前的报道一致,该突变体的50%最低抑菌浓度(MIC50>1.6μg/mL)远高于野生型菌株(MIC50≈0.034μg/mL)(见图3中B)。综上所述,该单碱基编辑***能够有效地在结核分枝杆菌中产生G:C到A:T碱基对替换,并能有效地表征基因的功能。
SEQUENCE LISTING
<110> 中国医学科学院病原生物学研究所
<120> 一种应用于结核分枝杆菌的单碱基基因编辑***及方法
<130> WX2021-90-001
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1
Claims (10)
1.一种应用于结核分枝杆菌的单碱基基因编辑***,是一种双质粒的CRISPR-Cas辅助的胞嘧啶核苷脱氨编辑***,包括单碱基编辑器质粒和抑制DNA修复元件质粒,其中,所述单碱基编辑器质粒包含sgRNA作用元件和编码nCas9sth、胞嘧啶核苷脱氨酶及尿嘧啶DNA糖基化酶抑制子形成的融合蛋白的基因,所述抑制DNA修复元件质粒包含降低RecA依赖的同源重组的编码RecX的基因元件和降低NucS依赖的错配修复的编码NucSE107A的基因元件。
2.如权利要求1所述的单碱基基因编辑***,其特征在于,所述单碱基编辑器质粒是复制性穿梭载体,含有分枝杆菌复制起点oriM和大肠杆菌复制起点oriE。
3.如权利要求2所述的单碱基基因编辑***,其特征在于,所述单碱基编辑器质粒还含有sacB反向筛选标记。
4.如权利要求1所述的单碱基基因编辑***,其特征在于,所述单碱基编辑器质粒上编码nCas9sth的基因为来源于嗜热链球菌(Streptococcus thermophilus)的cas9sth1基因,其蛋白质序列的第九位天冬氨酸突变为丙氨酸。
5.如权利要求1所述的单碱基基因编辑***,其特征在于,所述单碱基编辑器质粒上编码胞嘧啶核苷脱氨酶的基因是APOBEC1基因,其优化序列如SEQ ID No:9所示;编码尿嘧啶DNA糖基化酶抑制子的基因是UGI基因,其优化序列如SEQ ID No:10所示。
6.如权利要求5所述的单碱基基因编辑***,其特征在于,所述单碱基编辑器质粒上编码的融合蛋白中,APOBEC1通过如SEQ ID No:16所示的16个氨基酸的连接子连接到nCas9sth的N端;UGI通过如SEQ ID No:17所示的4个氨基酸的连接子连接到nCas9sth的C端。
7.如权利要求1所述的单碱基基因编辑***,其特征在于,所述单碱基编辑器质粒上的融合蛋白的基因由诱导型启动子驱动表达;所述抑制DNA修复元件质粒上的编码RecX的基因和编码NucSE107A的基因由诱导型启动子驱动表达。
8.如权利要求1所述的单碱基基因编辑***,其特征在于,所述NucSE107A为NucS蛋白序列第107位谷氨酸突变为丙氨酸的突变体。
9.一种对结核分枝杆菌进行单碱基基因编辑的方法,利用权利要求1~8任一所述的单碱基基因编辑***进行如下操作:
1)用所述抑制DNA修复元件质粒转化结核分枝杆菌,并将转化子制备成感受态细胞;
2)将靶向目标基因片段的sgRNA序列连接到所述单碱基编辑器质粒上的sgRNA作用元件中,得到含有sgRNA序列的单碱基编辑器质粒;
3)用步骤2)制备的含有sgRNA序列的单碱基编辑器质粒转化步骤1)制备的感受态细胞,诱导相关元件表达进行基因编辑,对得到的转化子进行验证,得到目标基因片段被编辑的转化子。
10.如权利要求9所述的方法,其特征在于,所述sgRNA序列长度为20bp,编辑目标位点位于目标基因片段中PAM序列远端第2-16位。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115725631A (zh) * | 2022-07-11 | 2023-03-03 | 中国科学院天津工业生物技术研究所 | 可控高突变率谷氨酸棒杆菌工程菌构建及在抗逆育种中应用 |
CN116751799A (zh) * | 2023-06-14 | 2023-09-15 | 江南大学 | 一种多位点双重碱基编辑器及其应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111850025A (zh) * | 2019-04-26 | 2020-10-30 | 中国医学科学院病原生物学研究所 | 一种应用于结核分枝杆菌的基因编辑***及方法 |
WO2021175287A1 (zh) * | 2020-03-04 | 2021-09-10 | 中国科学院遗传与发育生物学研究所 | 检测单碱基编辑***随机脱靶效应的方法 |
-
2021
- 2021-09-17 CN CN202111092318.3A patent/CN113774077A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111850025A (zh) * | 2019-04-26 | 2020-10-30 | 中国医学科学院病原生物学研究所 | 一种应用于结核分枝杆菌的基因编辑***及方法 |
WO2021175287A1 (zh) * | 2020-03-04 | 2021-09-10 | 中国科学院遗传与发育生物学研究所 | 检测单碱基编辑***随机脱靶效应的方法 |
Non-Patent Citations (2)
Title |
---|
DING X.Y.等: "Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase", 《FRONTIERS IN GENOME EDITING》 * |
李斯尚: "分枝杆菌基因组中单碱基基因编辑的研究", 《中国优秀硕士学位论文全文数据库 基础科学 医药卫生科技专辑》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115725631A (zh) * | 2022-07-11 | 2023-03-03 | 中国科学院天津工业生物技术研究所 | 可控高突变率谷氨酸棒杆菌工程菌构建及在抗逆育种中应用 |
CN116751799A (zh) * | 2023-06-14 | 2023-09-15 | 江南大学 | 一种多位点双重碱基编辑器及其应用 |
CN116751799B (zh) * | 2023-06-14 | 2024-01-26 | 江南大学 | 一种多位点双重碱基编辑器及其应用 |
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