CN109680053A - 新型SsCas12a蛋白在核酸检测方面的应用 - Google Patents
新型SsCas12a蛋白在核酸检测方面的应用 Download PDFInfo
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- CN109680053A CN109680053A CN201811519842.2A CN201811519842A CN109680053A CN 109680053 A CN109680053 A CN 109680053A CN 201811519842 A CN201811519842 A CN 201811519842A CN 109680053 A CN109680053 A CN 109680053A
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Abstract
本发明公开了新型SsCas12a蛋白在核酸检测方面的应用。本发明首次研究发现SsCas12a蛋白具有DNA切割活性,因此可作为新型CRISPR/SsCas12a系统应用于核酸检测、基因编辑、基因修饰,为基于Cas12a的基因编辑、修饰及分子检测提供了一种新的必要工具。同时本发明提供了一种包括SsCas12a蛋白和gRNA的核酸检测系统和试剂盒,可在25℃‑37℃的室温下实现高灵敏、高精度的分子检测,检测特异性好、灵敏度高,成本低廉、操作方便、快捷、应用范围广,在核酸检测方面具有很好的应用前景。
Description
技术领域
本发明属于分子生物学技术领域。更具体地,涉及新型SsCas12a蛋白在核酸检测方面的应用。
背景技术
为实现病原体等的高灵敏、高精度检测,开发低成本、准确、高效、快速的诊断方法非常重要。在病原体检测方面,使用经典的基于培养的表型试验测定易感性或抗性、ELISA等是临床病原体检测中使用的一般方法。但是由于病原体并不完全能在体外进行培养,其相关蛋白、小分子等检测灵敏度有限,传统的检测方法耗时并且准确性不高。而基于分子的诊断方法可以提高检测的速度和准确性,这对医院和社区环境中的感染控制、预防、治疗很有意义。
目前用于如病原体检测等的技术有:(1)分离培养技术:病原体分离培养,特别是对于病原微生物和病毒的分离培养,是早期的病原体金标准鉴定技术。该方法存在对的问题主要有:分离培养耗时长,往往需要几天时间,无法实现短时间内迅速得到检测结果,且必须高度依赖检测实验室硬件及实验操作人员条件,且不适用于目前未有成熟培养手段的病原微生物和病毒的检测。(2)免疫学检测:以基于抗原-抗体的免疫反应,识别病原相关蛋白,从蛋白水平对病原体进行检测。该方法存在检测灵敏度较低,且特异性受环境等影响较大,检测的窗口期较长,无法满足诊疗需求,仅适用于初筛而不能作为及时确诊的依据,不能够识别同一类病原的不同亚型等问题。(3)聚合酶链反应(PCR):包括普通PCR、等位基因特异性PCR、实时荧光定量PCR、PCR-Sanger测序技术、PCR-基因芯片技术等。PCR是从核酸水平对病原体进行检测,整个实验需要1~2小时完成。该方法的主要缺点在于进行PCR检测时需要依赖PCR仪或昂贵的实时定量PCR仪,及其它多种配套设备,以及专门的PCR实验室和专业操作人员。PCR检测无法实现即时检验、床旁诊断和无特定实验室检测条件的场景应用,因此无法满足基层、用户终端、现场的检验需求。同时,PCR检测可能存在假阳性和灵敏度不足等问题。
CRISPR/Cas系统可以识别出外源DNA或RNA,并将它们切断,沉默外源基因的表达。正是由于这种精确的靶向功能,CRISPR/Cas系统被开发成一种高效的基因编辑工具,可以对基因进行定点的精确编辑。CRISPR/Cas在向导RNA(guide RNA,gRNA)和Cas9蛋白的参与下,待编辑的细胞基因组DNA将被看作病毒或外源DNA,被精确剪切。CRISPR/Cas9的应用也有一些限制条件。首先,待编辑的区域附近需要存在相对保守的PAM序列(NGG)。其次,向导RNA要与PAM上游的序列碱基互补配对。再者,gRNA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gRNA,也是CRISPR-Cas9基因敲除的关键技术。
2015年,一种全新的第二类CRISPR-Cas系统-V型系统被发现,该系统中的效应蛋白被命名为Cpf1/Cas12a。张锋团队于2015年11月22日在《Cell》发表的一篇题为“Cpf1is asingle RNA-guided endonuclease ofa Class 2CRISPR-Cas system”的文章。该系统基本的工作流程与CRISPR/Cas9类似,还是借助CRISPR序列的“黑名单”系统对入侵者进行打击。但是gRNA形成的方式与CRISPR/Cas9系统不同:Cpf1/Cas12a蛋白会与未成熟的gRNA复合,并对gRNA进行加工,gRNA则会与PAM附近的互补区域杂交。最后,外源双链DNA(doublestrand DNA,dsDNA)会被剪切,其基因表达也会被沉默。然而,在切割靶dsDNA的同时,激活了的Cpf1/Cas12a还会降解靶dsDNA邻近的单链DNA(single strand DNA,ssDNA),称之为“附属切割”,这种活性是新开发的核酸检测平台的关键特征。2018年4月27日,Doudna团队和张锋团队同时在《Science》发表了两篇题为“Two distinct RNase activities ofCRISPR-C2c2enable guide-RNA processing and RNA detection”和“Multiplexed andportable nucleic acid detection platform with Cas13,Cas12a,and Csm6”的论文。表明Cpf1/Cas12a在切割靶dsDNA的同时,还会降解靶dsDNA邻近的ssDNA。这两个独立的实验室分别改造了靶向dsDNA的V型CRISPR系统,使其成为了快速、便宜且高度灵敏的诊断工具。这一发现有望为科学研究以及全球公共卫生带来变革性的影响。利用这种新的CRISPR技术:CRISPR-Cpf1/Cas12a,可以高灵敏度检测包括寨卡病毒感染,登革热病毒感染等在内的疾病,其原理在于将CRISPR-Cpf1/Cas12a与等温核酸扩增结合,检测特异性的RNA或DNA。另外,该系统还包含有一个切割时发出荧光的报告ssDNA。当Cpf1/Cas12a检测到靶标dsDNA序列时,其ssDNase活性就会切割报告ssDNA,释放可检测的荧光信号。将这两种技术结合起来的新系统能够以极低浓度检测单RNA和单DNA分子,具有很好的应用前景。另外根据张锋团队的研究结果显示,Cpf1/Cas12a蛋白同族间具有较大的差异,某些同族蛋白无活性。
发明内容
本发明要解决的技术问题是克服现有病原体等分子检测技术存在的缺陷和不足,提供一种具有DNA切割活性的新型CRISPR/Cas12a系统的蛋白组分SsCas12a,可应用于特异性核酸检测。
本发明的目的是提供SsCas12a蛋白在切割DNA方面以及核酸检测方面的应用。
本发明另一目的是提供一种基于SsCas12a蛋白的核酸检测系统。
本发明再一目的是提供一种基于新型CRISPR/SsCas12a系统的核酸检测方法。
本发明上述目的通过以下技术方案实现:
本发明首次研究发现SsCas12a蛋白具有DNA切割活性,具有靶向体外DNA序列或体内基因组序列进行特异切割的活性,因此可作为新型CRISPR/SsCas12a系统应用于核酸检测、基因编辑及基因修饰。SsCas12a的核苷酸序列如SEQ ID NO.1或SEQ ID NO.6所示,其来源菌种为Smithella sp.SCADC。同时发现,设计制备出精确、特异性靶向目标基因的gRNA等,是实现高灵敏、高精度核酸检测的关键。
因此,以下应用均应在本发明的范围之内:
SsCas12a蛋白在切割DNA方面的应用。
SsCas12a蛋白在作为或制备DNA切割工具方面的应用。
SsCas12a蛋白在核酸检测方面的应用。
SsCas12a蛋白在基于CRISPR/Cas12a的核酸检测方面的应用。
SsCas12a蛋白在作为或制备核酸检测工具方面的应用。
SsCas12a蛋白在作为或制备基于CRISPR/Cas12a的核酸检测工具方面的应用。
所述SsCas12a蛋白具有靶向体外DNA序列或体内基因组序列进行特异切割的活性,具体是gRNA介导的DNA切割活性。
另外基于上述应用,本发明还提供一种基于CRISPR/Cas12a的核酸检测系统,包括SsCas12a蛋白和gRNA。
其中,所述gRNA的设计原则为:在选取gRNA靶向序列时,靶向序列5’端应具有5’-TTTN-3’序列,且靶向序列本身不形成稳定二级结构、靶向序列和其余序列间不形成稳定二级结构。
作为一种可选择的案例,当靶序列如SEQ ID NO.2所示时,gRNA的序列如SEQ IDNO.3所示。
一种基于CRISPR/Cas12a的核酸检测方法,是利用SsCas12a蛋白和对应靶标序列的gRNA进行CRISPR核酸检测。
具体地,所述基于CRISPR/Cas12a的核酸检测方法为:将待测核酸样品、SsCas12a蛋白、gRNA、非特异单链荧光探针和反应所需缓冲液混合为反应体系,进行检测反应。具体是反应体系置于荧光分析仪(BioTek)进行荧光分析,以激发波长530nm/发射波长580nm读取反应孔荧光值。
优选地,检测体系包括:2μl RPA产物,45nM SsCas12a,22.5nM gRNA,100nM非特异单链DNA荧光探针,及核酸酶检测缓冲液。
所述缓冲液的各组分在检测体系的终浓度为:20mM Tris,60mM NaCl,10mMMgCl2,pH 7.3。
反应条件为:37℃下反应1-3小时。
同时,在上述应用和检测系统中,除了本发明所述的SsCas12a蛋白本身,其功能变体或其同源物或直向同源物皆有可能保留部分或全部蛋白质活性,即SsCas12a蛋白的功能变体或其同源物或直向同源物的应用,也应在本发明的范围之内。
所述功能变体可以包括ssCas12a突变体(可以是插入,缺失或替换序列的突变体)、多晶型等。所述功能变体也包括ssCas12a蛋白与另一种通常不相关的核酸、蛋白质或多肽的融合产物。功能变体可以是天然存在的,也可以是人造的。
另外具体地,本发明还提供了所述SsCas12a蛋白的制备方法。是先将SsCas12a蛋白基因序列经过密码子优化,优化后的SsCas12a蛋白基因序列如SEQ ID NO.6所示,使基因更适合在哺乳动物细胞中表达,然后将优化后的SsCas12a蛋白基因克隆入pET28a质粒,进行原核表达,表达菌采用BL21star(DE3);最后经过纯化得到SsCas12a蛋白。
优选地,蛋白制备条件如下:
本发明制备SsCas12a蛋白时的诱导表达条件为:按照1:80-120(优选1:100)的接种量,将表达菌接种LB培养基培养,于28-38℃(优选37℃)过夜培养菌种至OD600=0.6-1.0,冰上冷却(冷却时间优选30分钟)后,加终浓度为0.2mM-0.5mM的IPTG,于16-37℃继续培养4-24小时。
本发明纯化蛋白时,使用40-60mM Tris-HCl、pH7.5-8.5280-320mM NaCl、4-6%甘油和20-40mM咪唑组成的裂解缓冲液进行菌体裂解。
优选地,裂解缓冲液的组成为:50mM Tris-HCl、pH8.0300mM NaCl、5%甘油和30mM咪唑。
本发明纯化蛋白时,以Ni Sepharose FF柱进行纯化。
本发明纯化蛋白时,以40-60mM Tris-HCl、pH7.5-8.5280-320mM NaCl、8-12%甘油和240-260mM咪唑组成的洗脱缓冲液进行洗脱。
优选地,洗脱缓冲液的组成为:50mM Tris-HCl、pH8.0300mM NaCl、10%甘油和250mM咪唑。
本发明纯化蛋白时,以40-60mM Tris-HCl、pH7.5-8.5280-320mM NaCl和4-6%甘油组成的透析液进行透析。
优选地,透析液的组成为:50mM Tris-HCl、pH8.0300mM NaCl和5%甘油。
优选地,透析使用30kDa透析袋进行。
具体地,作为一种优选的可选择方案,本发明所述SsCas12a蛋白的制备方法包括如下步骤:
S1.以携带有SsCas12a序列的质粒为模版,利用SEQ ID NO.4和5所示上下游引物进行PCR扩增;
S2.利用NotI和NotI双酶切,将PCR扩增产物连接至pET28a-ccdB-CmR载体,得到重组质粒pET-28a-SsCas12a;
S3.重组质粒pET-28a-SsCas12a转化至BL21表达菌株,菌株用终浓度为0.2mM的IPTG,于18℃诱导表达24h;
S4.纯化:利用Ni Sepharose FF进行纯化;
S5.浓缩:纯化后的SsCas12a蛋白再以30kDa超滤管浓缩,得到的SsCas12a蛋白添加甘油至终浓度50%后,分装液氮速冻保存于-80℃。
其中,步骤S2所述pET-28-ccdB-CmR载体是以原核表达载体pET28a基础,在HindIII和XhoI酶切位点之间插入NotI-ccdB-CmR-AscI序列得到。
所述稀释使用的稀释液包含50mM Tris-HCl、pH8.0300mMNaCl、5%甘油;
所述透析液包含50mM Tris-HCl、pH8.0300mM NaCl、5%甘油。
步骤S1所述携带有SsCas12a序列的质粒是将包括C端SV40核定位信号和HA标签的SsCas12a蛋白,通过NotI与AscI酶切位点连于改良pET28a质粒获得;所述改良pET28a质粒是在原始pET28a质粒的基础上,在HindIII与XhoI之间,插入了NotI限制性核酸内切酶位点、氯霉素抗性基因、ccdB细菌生长致死蛋白、AscI限制性核酸内切酶位点获得。
经过本发明上述原核表达的SsCas12a蛋白,为来源Smithella sp.SCADC的CRISPR/SsCas12a系统的蛋白组分,其C端连有SV40核定位序列,能与体外转录的sgRNA结合,具有靶向体外DNA序列或体内基因组序列进行特异切割的活性。
另外,基于本发明的上述技术方案,应当涵盖如下内容:
(1)本发明通过SsCas12a附属切割带荧光基团与粹灭基团的报告DNA链,释放荧光基团,然后将反应体系置于荧光分析仪中检测核酸产物。除本发明所述的核酸检测信号报告方法,本发明也可通过其他利用激活SsCas12a产生附属切割效应后,实现信号检测的方案来检测样本中存在的一种或多种靶分子。
(2)在(1)中所述方案,包括基于SsCas12a附属切割带生物素、荧光基团、地高辛或其他标记的核酸片段后,通过胶体金侧向层析方式实现核酸产物检测。本发明也可通过SsCas12a附属切割聚集化的胶体金颗粒,使胶体金颗粒颜色变化,从而检测核酸产物信号。
(3)本发明在其他特异性核酸检测方案中,一种或多种指导RNA可被设计为靶向一种或多种诊断疾病状态的靶分子。所述疾病,可以是人类疾病,动物疾病,植物疾病;
(4)根据(3),所述人类疾病,可以是人类感染性疾病,癌症,器官疾病,血液疾病,免疫系统疾病,脑和神经系统疾病,内分泌疾病,遗传性疾病。
(5)根据(4),所述人类感染性疾病,可以是由病毒、细菌或真菌引起。可以是呼吸道合胞病毒、甲型流感病毒、乙型流感病毒、季节性流感病毒、副流感病毒、腺病毒、人鼻病毒、人偏肺病毒、腮腺炎病毒、肺炎支原体、肺炎衣原体、结核分枝杆菌、中东呼吸综合征冠状病毒、百日咳杆菌、嗜肺军团杆菌、A链球菌;可以是人类免疫缺陷病毒、淋球菌、沙眼衣原体、解脲脲原体、人乳头瘤病毒、梅毒螺旋体、单纯疱疹病毒、人细小病毒;可以是甲型肝炎病毒、乙型肝炎病毒、丙型肝炎病毒、丁型肝炎病毒、戊型肝炎病毒;可以是人巨细胞病毒、人疱疹病毒、柯萨奇病毒、肠道病毒EV71/CA16、登革热病毒、沙门氏菌、志贺氏菌、幽门螺旋杆菌、诺如病毒、肠道腺病毒、轮状病毒、埃博拉病毒。登革热病毒。
(6)根据(4),癌症可以是肺癌、结直肠癌、胃癌、胃肠间质瘤、乳腺癌、卵巢癌、前列腺癌、甲状腺癌、胰腺癌、淋巴瘤等。
(7)根据(4),血液疾病及遗传性疾病可以是:地中海贫血、血友病、镰刀型细胞贫血、Rett综合征、囊性纤维化、亨廷顿病、脆性X综合征、13三体综合征、18三体综合征、21三体综合征、遗传性代谢疾病、遗传性耳聋、遗传多囊肾病、先天性糖基化病、G6PD缺乏症、苯丙酮尿症、酪氨酸血症、肝豆状核变性、白化病、糖原累积病、遗传性乳腺癌、遗传性卵巢癌、遗传性结直肠癌等;
(8)根据(4),所述器官疾病,免疫系统疾病,脑和神经系统疾病,内分泌疾病,可以是脑卒中、高血压、冠心病、肌萎缩侧索硬化症、帕金森病、阿尔兹海默病、过敏性疾病、类风湿、多发性硬化症、遗传性过敏性皮炎、糖尿病、黄斑变性、强直性脊柱炎等。
(9)根据(3),所述动物疾病可以是:猪流行性腹泻病毒、猪轮状病毒A群、猪传染性胃肠炎病毒、口蹄疫、蓝耳病、猪瘟、猪圆环病毒、非洲猪瘟、猪伪狂犬病毒、猪日本乙型脑炎、猪细小病毒、猪流感、蓝耳病、猪链球菌、猪丹毒杆菌、牛瘟、小反刍兽疫病毒、绵羊痘病毒、多杀性巴氏杆菌、禽流感、新城疫病毒、鸭瘟病毒、鸡马立克氏病病毒、鸡传染性法氏囊病毒、猫衣原体、猫冠状病毒、猫支原体、猫传染性腹膜炎、猫杯状病毒、猫疱疹病毒、猫泛白细胞减少症、犬支原体、犬腺病毒、犬副流感、犬甲型流感、犬细小病毒、犬瘟热病毒、犬冠状病毒、狂犬病毒、巴尔通体、弓形虫、钩端螺旋体、巴贝斯虫、布氏杆菌、对虾传染性肌肉坏死病毒、对虾黄头病病毒、对虾淘拉综合症病毒、对虾传染性皮下及造血组织坏死病毒、炭疽杆菌等。
(10)本发明在其他特异性核酸检测方案中,一种或多种指导RNA可被设计为靶向一种或多种微生物抗性基因。所述抗性基因,可以是四环素耐药、氨基糖苷类药物耐药、耐消毒剂、红霉素耐药、大环内酯外排、万古霉素耐药、多药耐药外排泵、莫匹罗星耐药、磺胺类耐药、泰洛星耐药、氟喹诺酮耐药、beta内酰胺酶类耐药、头孢菌素耐药、碳青霉烯酶耐药、金黄色葡萄球菌耐药、氯霉素酰基转移酶基因、博来霉素基因、嘌呤霉素基因、卡那霉素基因、氨苄霉素基因、超广谱β-内酰胺酶耐药基因等。
(11)本发明在其他特异性核酸检测方案中,一种或多种指导RNA可被设计为靶向一种或多种个体基因型的靶分子。所述个体基因型,可以是人类单核苷酸多态性及基因型,动物基因型,植物基因型等。
(12)根据(11),所述人类单核苷酸多态性及基因型可以是疾病相关多态性位点,包括VKORC1、CYP2C9、CYP2C19等;可以是性状相关多态性位点,包括乳糖耐受基因、咖啡因代谢、酒精代谢、皮肤抗氧化、味觉敏感度、脱发等;可以是人类白细胞抗原(HLA);
(13)根据(11),所述动物基因型,植物基因型,可以是单核苷酸多态性、等位基因、育种鉴定、转基因鉴定等。
(14)本发明在其他特异性核酸检测方案中,一种或多种指导RNA可被设计为靶向一种或多种检测环境样本状态的靶分子。所述环境样品来自食品样品,饮料样品,纸张表面,织物表面,金属表面,木材表面,塑料表面,土壤样品,水样品,大气或其他气体样品,或其组合。所述检测环境样本状态,可以是病毒,细菌,真菌等各类微生物核酸存在状态,也可以是动物,植物基因组来源核酸存在状态,也可以是转基因核酸存在状态;
(15)本发明在其他特异性核酸检测方案中,一种或多种样本类型可用于核酸检测。所述样本类型,可以是组织样本,唾液,血液,血浆,血清,粪便,尿液,痰液,粘液,淋巴液,滑液,脑脊液,腹水,胸腔积液,血清肿,脓或皮肤或粘膜表面的拭子,盥洗液等;
(16)本发明在其他特异性核酸检测方案中,该核酸检测反应可以承载于不同基质上;所述基质,可以是试管,液滴,固体腔路,微孔,特定基质(如纸基质)等。
本发明具有以下有益效果:
本发明研究探索寻找到具有DNA切割活性的SsCas12a蛋白,具有靶向体外DNA序列或体内基因组序列进行特异切割的活性,可作为新型CRISPR/SsCas12a系统应用于核酸检测,为基于CRISPR/Cas12a系统的核酸检测提供了一种新的选择。
而且基于SsCas12a蛋白的核酸检测系统可在25-37℃实现高灵敏、高精度的分子检测,具有很好的特异性和兼容性,检测灵敏度高,且检测成本低廉、操作方便、快捷、应用范围广,在核酸检测方面具有很好的应用前景。
附图说明
图1为SsCas12a片段PCR扩增结果。
图2为重组质粒pET28a-SsCas12a载体酶切鉴定结果。
图3为SsCas12a蛋白表达结果。
图4为SsCas12a蛋白纯化结果。
图5为SsCas12a活性检测结果;1、阴性对照;2、实验组。
图6为SsCas12a gRNA纯化结果。
图7为基于SsCas12a的核酸检测结果。
图8为SsCas12a核酸检测灵敏度检测结果(黑色为LbCas12a,灰色为SsCas12a)。
图9为SsCas12a核酸检测特异性检测结果。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。对于本领域技术人员来说,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明,以下实施例所用试剂和材料均为市购。
除非另有说明,本发明采用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等是本领域的常规技能。参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis),《分子克隆:实验室手册》(MOLECULAR CLONING:ALABORATORY MANUAL),第2次编辑(1989);《当代分子生物学实验手册》(CURRENT PROTOCOLSIN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑,(1987));《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司):《PCR2:实用方法》(PCR 2:A PRACTICALAPPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))、哈洛(Harlow)和拉内(Lane)编辑(1988)《抗体:实验室手册》(ANTIBODIES,A LABORATORY MANUAL),以及《动物细胞培养》(ANIMALCELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
如下述实施例中所用的异丙基硫化-D-半乳糖苷(IPTG)购买Sigma公司。NiSepharose FF购买自GE Healthcare。蛋白纯化耗材购买自碧云天公司。Amicon 430kDa超滤管购买自Millipore公司。Phusion DNA聚合酶、FastDigestNotI、FastDigestAscI内切酶、T4连接酶购买自Thermo公司。PCR clean up和凝胶回收试剂盒均购买自Qiagen公司。
实施例1 SsCas12a蛋白的活性检测
一、我们研究意外发现,SsCas12a蛋白可在gRNA介导下被实现双链DNA底物的特异切割。然而在张锋团队于2015年11月22日在《Cell》发表的一篇题为“Cpf1is a singleRNA-guided endonuclease of a Class 2CRISPR-Cas system”的文章中报道,与SsCas12a蛋白序列相同的一个蛋白无DNA切割活性。
鉴于此种情况,本发明人团队又做了大量的探索和验证工作,最终证实了SsCas12a的DNA切割活性,作为CRISPR/SsCas12a系统应用于核酸检测具有很好的特异性和灵敏性。同时,本发明人团队研究发现,在选取gRNA靶向序列时,靶向序列5’端应具有5’-TTTN-3’序列,且靶向序列本身不形成稳定二级结构、靶向序列和其余序列间不形成稳定二级结构,在此sgRNA设计原则下,SsCas12a具有靶向体外DNA序列或体内基因组序列进行特异切割的活性。
以下给出了SsCas12a蛋白的制备以及活性验证实验案例。
二、Cpf1/SsCas12a基因的克隆和蛋白表达
采用源自Smithella sp.SCADC的Cas12a蛋白基因,经过密码子优化,使基因更适合在哺乳动物细胞中表达。优化后的Cas12a蛋白基因克隆入pET28a质粒,进行原核表达。表达菌采用BL21star(DE3)。将Cas12a蛋白重组表达载体转化后,进行蛋白表达、SDS-PAGE检测以及凝胶柱纯化,获得的纯化后的Cas12a蛋白放-80℃保存。具体方法如下:
1、Cpf1/SsCas12a基因的克隆和蛋白表达
(1)PCR扩增Cas12a序列
a.设计引物
根据SsCas12a序列设计了上下游引物,序列如下:
上游引物(SEQ ID NO.4所示):
GTGCGGCCGCCATGCAGACCCTGTTTGAGAACTTCACA;
下游引物(SEQ ID NO.5所示):
GTGGCGCGCCTGGCATAGTCGGGGACATCATATG。
b.PCR扩增
先将SsCas12a蛋白基因进行优化,优化后序列如SEQ ID NO.6所示。利用上述上下游引物,使用高保真DNA聚合酶(phusion DNA聚合酶)在不同退火温度下对目的片段进行PCR扩增。结果如附图1所示,PCR目的条带(约4000bp)。
(2)构建重组质粒pET-28a-SsCas12a
a.PCR扩增产物纯化:PCR扩增后产物通过Qiagen公司的纯化试剂盒(Cleanup试剂盒)进行纯化处理;
b.使用Thermo公司的快速限制性内切酶NotI(FastDigestNotI)和NotI(FastDigestAscI)进行双酶切;
c.酶切产物通过Qiagen公司的微量样品凝胶回收试剂盒(MiniElute)纯化回收;
d.纯化回收的产物连接至同样经过NotI和AscI双酶切处理的pET28a-ccdB-CmR载体上,得到重组质粒pET-28a-SsCas12a;
其中,所用pET-28-ccdB-CmR载体为本实验室保存,是以原核表达载体pET28a(购于生物通代理)基础,经改造在HindIII和XhoI酶切位点之间添加NotI-ccdB-CmR-AscI序列,制成pET-28-ccdB-CmR载体。
(3)重组质粒pET-28a-SsCas12a的鉴定
为鉴定pET-28a-SsCas12a重组载体正确与否,我们对重组质粒pET-28a-SsCas12a进行酶切鉴定和测序鉴定。
分别使用Asc I或NotI单酶切和Asc I或NotI双酶切进行酶切鉴定,结果如附图2所示,实验结果表明,所有实验组的酶切产物大小均为与预期相符,因而可初步判断我们得到的载体为正确的pET-28a-SsCas12a载体。
另外,测序结果也表明SsCas12a序列被正确地克隆至pET28a。
(4)SsCas12a蛋白的原核表达
a.将鉴定正确的重组质粒pET-28a-SsCas12a转化至BL21(DE3)表达菌株(购于Transgen公司)中。经过阳性鉴定获得重组菌。
b.挑取重组菌的单克隆至50mL LB培养基中37℃培养过夜。按照1:100的接种量,将过夜菌种接种至1L LB培养基中37℃培养至OD600=1.0,冰水浴30min,加IPTG至终浓度为0.2mM,于15-37℃继续培养24h。离心收集菌体于-80℃保存。
c.检测并优化SsCas12a蛋白表达
将重组质粒pET-28a-SsCas12a转入BL21(DE3)中,在37℃以0.2mM诱导蛋白表达,并对裂解后沉淀和上清进行电泳分析。(如附图3所示)。
2、SsCas12a蛋白的纯化
(1)纯化方法
诱导表达后的菌液进行离心,将菌体重悬于裂解缓冲液中,进行超声破碎(70%振幅,2s On/4s Off,3分钟,Sonics 750w超声仪),离心分离上清液。将蛋白裂解上清上样至平衡后的Ni Sepharose FF,以大于30倍柱床体积的裂解缓冲液洗去杂蛋白,以洗脱缓冲液进行洗脱,以Superdex 200,Tricorn 10/300凝胶色谱柱进行纯化。SDS-PAGE分析观察结果以及凝胶柱纯化,获得的纯化后的Cas12a蛋白。其中,裂解缓冲液包含50mM Tris-HCl、pH8.0300mM NaCl、5%甘油、20mM咪唑。洗脱缓冲液包含50mM Tris-HCl、pH8.0300mM NaCl、5%甘油、250mM咪唑。
得到的蛋白用三倍稀释的透析液以30kDa超滤管或透析袋浓缩(所用透析液包括50mM Tris-HCl、pH8.0300mM NaCl、5%甘油)。添加甘油至终浓度50%后,分装液氮速冻保存于-80℃。
(2)纯化结果
在优化纯化步骤后再次进行大量纯化,目的条带约为170kDa。如附图4所示,可见纯化的纯度及产量较高。
本发明SsCas12a纯化方案简化了TEV切割标签步骤,大幅精简了纯化流程和纯化成本,同时,该方法能确保蛋白活性。
三、SsCas12a蛋白的活性检测
1、为检测SsCas12a蛋白的活性,通过体外实验验证纯化的SsCas12a的活性。
我们准备了含靶标序列的pUC19-B2质粒(质粒来源于文章:CRISPR-Cas12atargetbinding unleashes single-stranded DNase activity)作为检验SsCas12a蛋白活性的底物,选取质粒上的一个位点设计sgRNA,有活性的SsCas12a蛋白与sgRNA结合后可将靶向DNA切割成两个片段,从而确定SsCas12a蛋白有活性与否。
具体是将100ng经SacI线性化的pUC19-B2质粒,20ng sgRNA,250ng纯化后的SsCas12a蛋白,混匀于1×NEB buffer3终体积10μL,37℃孵育60min,反应以0.1μL蛋白酶K终止,在1%琼脂糖凝胶中进行电泳分析SsCas12a蛋白体外活性。
实验中所述gRNA的获得:设计能识别质粒的靶向序列:tttaGATCGTTACGCTAACTATGA,通过T7RNA聚合酶转录出sgRNA。
2、结果如附图5所示,只有当添加了SsCas12a蛋白后DNA才可被成功切割成两个片段,从而证实我们所提取、纯化的SsCas12a蛋白在体外有活性。
实施例2基于SsCas12a的核酸检测
1、制备靶RNA
靶核苷酸可经过PCR扩增、重组酶聚合酶扩增(RPA),NASBA等温扩增或、环介导等温扩增(LAMP)、链置换扩增(SDA)、解旋酶依赖性扩增(HDA)和切口酶扩增反应(NEAR)方式扩增靶DNA。
重组酶聚合酶扩增RPA(Recombinase Polymerase Amplification):采用NCBIPrimer blast设计RPA引物,扩增片段大小为80-120nt,引物的变性温度可为54-67℃、Opt=60,长度为30-35nt、Opt=32,引物中GC含量为40-60%,根据设计序列合成DNA引物。
模版序列:
atttcacacaggaaacagctatgaccatgattacgccaagcttGACGACAAAAtttaGATCGTTACGCTAACTATGAgggctgtctgtgaatgctaggatccccgggtaccgagctcgaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcc
引物序列:
FP:AATTCTAATACGACTCACTATAGggccagtgaattcgagctcggtacccggggatcc
RP:atttcACACAGGAAACAGCTATGACCATGATTACG
分别参考Basic和BasicRT(TwistDx)试剂盒进行RPA反应,不同的是,在模板片段加入之前,先加入280mM的MgAc,即乙酸镁。在37℃下反应30分钟。
胶分离以及纯化(采用MinElute gel extraction kit(Qiagen)试剂盒),纯化后的dsDNA与T7聚合酶37℃孵育过夜(采用T7RNApolymerase(Thermo)试剂盒),然后用RNeasymini kit(Qiagen)试剂盒纯化RNA,从而获得靶核RNA。
2、制备gRNA
gRNA引物序列设计原则:选取靶向序列时,靶向序列5’端应具有5’-TTTN-3’序列;且靶向序列本身不形成稳定二级结构、靶向序列和其余序列间不形成稳定二级结构。可通过http://www.rgenome.net/cas-designer/在线软件辅助设计。
引物结构:
5’-靶向序列-“ATCTACACTTAGTAGAAATTA”-CCCTATAGTGAGTCGTATTACA-3’
其中“ATCTACACTTAGTAGAAATTA”序列可替换为“ATCTACAATAGTAGAAATTA”。
参照T7RNApolymerase kit(Thermo)试剂盒说明书,将带T7启动子的DNA片段、T7引物、T7聚合酶混合,37℃孵育过夜;再采用RNeasy mini kit(Qiagen),获得纯化的gRNAs。如图6所示。
T7引物序列:TGTAATACGACTCACTATAGGG
T7gRNA引物序列:
TCATAGTTAGCGTAACGATCATCTACACTTAGTAGAAATTACCCTATAGTGAGTCGTATTACA。
3、Cpf1/Cas12a活性检测
检测体系包括:100ng核酸底物,45nM纯化的SsCas12a,22.5nM gRNA,及核酸酶检测缓冲液(20mM Tris,60mM NaCl,10mM MgCl 2,pH 7.3)。反应1小时。加入1μl蛋白酶K(Thermo)终止反应。琼脂糖凝胶电泳分析结果,如图7。
4、核酸检测
检测体系包括:2μl RPA产物,45nM纯化的SsCas12a,22.5nM gRNA,100nM在SsCas12a切割时可发出荧光的报告DNA链,即非特异单链DNA荧光探针(DNAseAlert QCSystem Thermo Scientific),0.5μl RNase抑制剂(Promega),及核酸酶检测缓冲液(缓冲液各组分在检测体系的终浓度为:20mM Tris,60mMNaCl,10mM MgCl 2,pH 7.3)。反应体系置于荧光分析仪(BioTek),在37℃(除非另有说明)下反应1-3小时,荧光动力检测5分钟一次。
分析SHERLOCK荧光数据:为了计算去除背景的荧光数据,方便不同条件之间的比较,样品的初始荧光被去除。背景荧光(无靶核苷酸或无gRNA的条件下)会从样品中去除,从而获得扣除背景荧光的数据。
检测结果如图8所示,与已报道的LbCas12a相比,SsCas12a具有相同检测灵敏度。
实施例3基于SsCas12a的核酸检测特异性
1、实验方法
将同实施例4中所述的待测核酸样品、SsCas12a蛋白、gRNA、非特异单链荧光探针和反应所需缓冲液加入混合为反应体系,以激发波长530nm/发射波长580nm读取反应孔荧光值。在靶标(底物)存在的情况下,与阴性对照样品(突变底物)相比,580nm处可接收到荧光信号。
根据SsCas12a针对底物和突变底物荧光信号强度差异判断检测特异性。
底物序列:
atatgGTGCCATGGACTTTAGTACATTGCAAGATACTAAATGTGAGGTAC
突变底物序列:
atatgGTGCCATGGACTTTAAAACATTGCAAGATACTAAATGTGAGGTAC
2、结果如图9所示,能够对特异和非完全配对的非特异模版进行区分,具有很好的核实检测特异性。
本领域技术人员可以理解的是,可以采用本领域常规的替代方法替换本发明实施例中常规的Cas12a基因的克隆、重组表达载体的构建、Cas12a蛋白的表达及纯化、靶核苷酸/目标基因片段的扩增等步骤中的一种或多种,以期获得类似或等同的效果。
序列表
<110> 广州普世利华科技有限公司
<120> 新型SsCas12a蛋白在核酸检测方面的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3753
<212> DNA
<213> SsCas12a序列(SsCas12a)
<400> 1
atgcaaacac tatttgaaaa tttcacaaat cagtatccgg tgagtaagac tttgcgtttt 60
gagttgatcc cgcagggaaa aacaaaagat tttatagaac agaaaggtct tttaaaaaaa 120
gacgaggatc gtgcggaaaa atataagaaa gtaaaaaaca ttattgatga atatcataaa 180
gatttcattg agaaatcact gaacggttta aaattagacg ggcttgagga gtataagact 240
ttatatttaa agcaggaaaa agacgataaa gataaaaagg cgtttgataa agaaaaagaa 300
aatcttagga aacagattgc gaatgctttt agaaacaatg aaaagtttaa aacgctcttt 360
gccaaggaat taataaagaa cgatttgatg agttttgcct gtgaggaaga caaaaaaaac 420
gtaaaggagt ttgaagcttt tacaacttat ttcactgggt ttcaccagaa cagggcaaac 480
atgtatgtgg cggatgagaa acgaacggct attgcgagcc ggcttataca tgagaatctg 540
cccaagttca ttgataacat caagatattt gaaaagatga aaaaagaagc acccgagctt 600
ctttcccctt ttaatcaaac attaaaggat atgaaggatg ttatcaaggg tacaacattg 660
gaagaaatat tttcactgga ttatttcaat aagacactta cacaatcggg gattgacata 720
tacaattccg taatcggcgg cagaacaccc gaagagggaa aaaccaaaat caagggctta 780
aacgaatata tcaatactga ttttaaccag aagcagaccg ataagaaaaa aagacagccc 840
aagttcaagc aactctacaa gcagattttg agcgacaggc agagcctgtc atttattgcg 900
gaggcattca aaaatgatac tgaaattctg gaagcaatag aaaaattcta tgtaaatgaa 960
ttgctgcatt tttccaatga aggcaaatca accaatgtat tggatgcaat caaaaatgct 1020
gtaagcaatc ttgaatcttt caacctgact aagatttatt tcagaagcgg cacttcattg 1080
accgatgttt ccaggaaagt atttggagaa tggagcatta ttaacagggc tttagataat 1140
tactacgcaa caacctaccc catcaagcca agagagaaat cagagaaata cgaagagcgg 1200
aaagagaaat ggctcaaaca ggattttaac gtcagcctaa ttcagaccgc aattgatgag 1260
tacgacaatg aaacagttaa gggaaaaaac agtgggaaag tcatcgttga ttactttgcg 1320
aagttctgcg atgacaaaga gactgattta attcagaagg tcaatgaagg ctatatagct 1380
gtcaaggatt tgctgaacac cccttatcct gaaaatgaga agctcgggag caataaagat 1440
caggtcaaac agataaaggc atttatggac agcataatgg acattatgca ttttgtaaga 1500
ccgctgagcc tgaaggatac tgataaagag aaagacgaaa cattctatag tctgtttacc 1560
cctctatacg accatctgac ccagaccatc gcactctaca ataaggtgcg taactacctc 1620
acacagaaac catacagcac ggaaaagata aaactgaact ttgaaaactc cacattgctg 1680
ggtggatggg atttaaataa agaaacagac aatacagcta ttatactgag gaaagagaac 1740
ctttattatc tgggcattat ggacaagagg cataacagga tattcagaaa cgtacctaaa 1800
gcggataaaa aggattcctg ctacgagaaa atggtttaca agcttctgcc tggagcaaat 1860
aaaatgctgc cgaaggtatt cttttcgcag tcacggatac aggaatttac tccgtcagcc 1920
aaactgctgg aaaactacga gaatgagacg cacaaaaaag gtgataattt caacctgaat 1980
cattgccacc aattaattga ttttttcaaa gactcaatca acaaacacga ggactggaaa 2040
aactttgatt tcaggttttc agccacatcc acctatgctg atttaagcgg attctaccac 2100
gaggtggaac atcagggcta taagataagt ttccagagca tagccgattc cttcattgac 2160
gatttggtca atgaagggaa actctatctt ttccaaatat acaacaagga tttctcacct 2220
ttcagtaagg gcaagcccaa cctgcacacc ctctactgga agatgctctt tgatgagaat 2280
aatttaaaag acgtggttta taaactaaat ggtgaggccg aggtattcta ccgcaagaaa 2340
tccattgccg agaaaaacac gaccattcat aaggccaatg aaagtattat taacaagaat 2400
ccagacaatc cgaaagccac aagtacattt aactacgaca tcgtcaagga taaacgctac 2460
accattgata agttccaatt ccacgttccc ataaccatga atttcaaggc tgaaggcata 2520
ttcaatatga accagagggt caaccagttc ctcaaggcca acccagatat taatatcatc 2580
ggaatagaca ggggagaaag gcatctactt tactacaccc tgataaatca gaaaggtaag 2640
attctcaagc aggacacctt gaatgtcatt gccaatgaaa agcagaaagt tgactatcac 2700
aacctgctgg acaagaaaga gggtgataga gccacggcaa ggcaggaatg gggcgtaatt 2760
gagaccatta aggaactgaa ggaaggttat ctgtcgcagg tcatccacaa actgaccgat 2820
ttgatgattg aaaacaacgc catcattgtg atggaagatt tgaacttcgg tttcaagcgt 2880
ggaaggcaga aggtggagaa gcaggtttac cagaagtttg agaaaatgct gattgataaa 2940
ctcaattacc ttgtggataa gaataaaaaa gcaaatgaac ttggcggtct gctcaacgca 3000
ttccagttag cgaacaagtt tgaaagtttc cagaaaatgg ggaagcagaa cggatttatt 3060
ttctacgtgc ctgcgtggaa cacaagcaag actgaccctg ccacaggttt cattgatttc 3120
ctgaaaccta gatatgagaa cctgaaacag gcaaaggact tctttgagaa gtttgattcc 3180
atccgtctca acagcaaggc agattatttt gaatttgctt ttgattttaa aaacttcacc 3240
ggaaaggcag atggtggaag gacgaaatgg acagtttgca ccaccaatga ggacaggtac 3300
gcctggaaca gggcgttaaa caacaacagg ggcagtcagg aaaaatacga tatcacagca 3360
gaactgaaat ccctgtttga cggaaaggtg gactataaaa gcggcaagga tttgaaacag 3420
cagatagcaa gtcaggaatt ggccgatttc ttcaggacat taatgaaata tttaagtgtc 3480
acactttcat tgaggcacaa caacggagaa aaaggtgaga cagagcagga ttatattctt 3540
tcgcctgttg cagacagtat gggaaaattc tttgattcaa ggaaagcagg tgacgatatg 3600
cccaagaatg ccgacgccaa cggcgcttat catatcgctc ttaaaggttt atggtgtctg 3660
gaacagatca gtaagacgga cgacctgaaa aaagtaaagt tagccataag caacaaagaa 3720
tggcttgaat ttatgcaaac actgaaagga taa 3753
<210> 2
<211> 185
<212> DNA
<213> 模板靶序列(Target sequence)
<400> 2
atttcacaca ggaaacagct atgaccatga ttacgccaag cttgacgaca aaatttagat 60
cgttacgcta actatgaggg ctgtctgtga atgctaggat ccccgggtac cgagctcgaa 120
ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 180
tcgcc 185
<210> 3
<211> 40
<212> DNA
<213> gRNA序列(gRNA)
<400> 3
taatttctac tattgtagat gatcgttacg ctaactatga 40
<210> 4
<211> 38
<212> DNA
<213> 上游引物(forward primer)
<400> 4
gtgcggccgc catgcagacc ctgtttgaga acttcaca 38
<210> 5
<211> 34
<212> DNA
<213> 下游引物(reverse primer)
<400> 5
gtggcgcgcc tggcatagtc ggggacatca tatg 34
<210> 6
<211> 3885
<212> DNA
<213> 优化后的SsCas12a蛋白基因序列(Optimized SsCas12a)
<400> 6
atgcagaccc tgtttgagaa cttcacaaat cagtacccag tgtccaagac cctgcgcttt 60
gagctgatcc cccagggcaa gacaaaggac ttcatcgagc agaagggcct gctgaagaag 120
gatgaggacc gggccgagaa gtataagaag gtgaagaaca tcatcgatga gtaccacaag 180
gacttcatcg agaagtctct gaatggcctg aagctggacg gcctggagaa gtacaagacc 240
ctgtatctga agcaggagaa ggacgataag gataagaagg cctttgacaa ggagaaggag 300
aacctgcgca agcagatcgc caatgccttc cggaacaatg agaagtttaa gacactgttc 360
gccaaggagc tgatcaagaa cgatctgatg tctttcgcct gcgaggagga caagaagaat 420
gtgaaggagt ttgaggcctt caccacatac ttcaccggct tccaccagaa ccgcgccaat 480
atgtacgtgg ccgatgagaa gagaacagcc atcgccagca ggctgatcca cgagaacctg 540
ccaaagttta tcgacaatat caagatcttc gagaagatga agaaggaggc ccccgagctg 600
ctgtctcctt tcaaccagac cctgaaggat atgaaggacg tgatcaaggg caccacactg 660
gaggagatct ttagcctgga ttatttcaac aagaccctga cacagagcgg catcgacatc 720
tacaattccg tgatcggcgg cagaacccct gaggagggca agacaaagat caagggcctg 780
aacgagtaca tcaataccga cttcaaccag aagcagacag acaagaagaa gcggcagcca 840
aagttcaagc agctgtataa gcagatcctg agcgataggc agagcctgtc ctttatcgcc 900
gaggccttca agaacgacac cgagatcctg gaggccatcg agaagtttta cgtgaatgag 960
ctgctgcact tcagcaatga gggcaagtcc acaaacgtgc tggacgccat caagaatgcc 1020
gtgtctaacc tggagagctt taacctgacc aagatgtatt tccgctccgg cgcctctctg 1080
acagacgtga gccggaaggt gtttggcgag tggagcatca tcaatagagc cctggacaac 1140
tactatgcca ccacatatcc aatcaagccc agagagaagt ctgagaagta cgaggagagg 1200
aaggagaagt ggctgaagca ggacttcaac gtgagcctga tccagaccgc catcgatgag 1260
tacgacaacg agacagtgaa gggcaagaac agcggcaaag tgatcgccga ttattttgcc 1320
aagttctgcg acgataagga gacagacctg atccagaagg tgaacgaggg ctacatcgcc 1380
gtgaaggatc tgctgaatac accctgtcct gagaacgaga agctgggcag caataaggac 1440
caggtgaagc agatcaaggc ctttatggat tctatcatgg acatcatgca cttcgtgcgc 1500
cccctgagcc tgaaggatac cgacaaggag aaggatgaga cattctactc cctgttcaca 1560
cctctgtacg accacctgac ccagacaatc gccctgtata acaaggtgcg gaactatctg 1620
acccagaagc cttacagcac agagaagatc aagctgaact tcgagaacag caccctgctg 1680
ggcggctggg atctgaataa ggagacagac aacacagcca tcatcctgag gaaggataac 1740
ctgtactatc tgggcatcat ggacaagagg cacaatcgca tctttcggaa cgtgcccaag 1800
gccgataaga aggacttctg ctacgagaag atggtgtata agctgctgcc tggcgccaac 1860
aagatgctgc caaaggtgtt cttttctcag agcagaatcc aggagtttac cccttccgcc 1920
aagctgctgg agaactacgc caatgagaca cacaagaagg gcgataattt caacctgaat 1980
cactgtcaca agctgatcga tttctttaag gactctatca acaagcacga ggattggaag 2040
aatttcgact ttaggttcag cgccacctcc acctacgccg acctgagcgg cttttaccac 2100
gaggtggagc accagggcta caagatctct tttcagagcg tggccgattc cttcatcgac 2160
gatctggtga acgagggcaa gctgtacctg ttccagatct ataataagga cttttcccca 2220
ttctctaagg gcaagcccaa cctgcacacc ctgtactgga agatgctgtt tgatgagaac 2280
aatctgaagg acgtggtgta taagctgaat ggcgaggccg aggtgttcta ccgcaagaag 2340
agcattgccg agaagaacac cacaatccac aaggccaatg agtccatcat caacaagaat 2400
cctgataacc caaaggccac cagcaccttc aactatgata tcgtgaagga caagagatac 2460
accatcgaca agtttcagtt ccacatccca atcacaatga actttaaggc cgagggcatc 2520
ttcaacatga atcagagggt gaatcagttc ctgaaggcca atcccgatat caacatcatc 2580
ggcatcgaca gaggcgagag gcacctgctg tactatgccc tgatcaacca gaagggcaag 2640
atcctgaagc aggataccct gaatgtgatc gccaacgaga agcagaaggt ggactaccac 2700
aatctgctgg ataagaagga gggcgaccgc gcaaccgcaa ggcaggagtg gggcgtgatc 2760
gagacaatca aggagctgaa ggagggctat ctgtcccagg tcatccacaa gctgaccgat 2820
ctgatgatcg agaacaatgc catcatcgtg atggaggacc tgaactttgg cttcaagcgg 2880
ggcagacaga aggtggagaa gcaggtgtat cagaagtttg agaagatgct gatcgataag 2940
ctgaattacc tggtggacaa gaataagaag gcaaacgagc tgggaggcct gctgaacgca 3000
ttccagctgg ccaataagtt tgagtccttc cagaagatgg gcaagcagaa cggctttatc 3060
ttctacgtgc ccgcctggaa tacctctaag acagatcctg ccaccggctt tatcgacttc 3120
ctgaagcccc gctatgagaa cctgaatcag gccaaggatt tctttgagaa gtttgactct 3180
atccggctga acagcaaggc cgattacttt gagttcgcct ttgacttcaa gaatttcacc 3240
gagaaggccg atggcggcag aaccaagtgg acagtgtgca ccacaaacga ggacagatat 3300
gcctggaata gggccctgaa caataacagg ggcagccagg agaagtacga catcacagcc 3360
gagctgaagt ccctgttcga tggcaaggtg gactataagt ctggcaagga tctgaagcag 3420
cagatcgcca gccaggagtc cgccgacttc tttaaggccc tgatgaagaa cctgtccatc 3480
accctgtctc tgagacacaa taacggcgag aagggcgata atgagcagga ctacatcctg 3540
tcccctgtgg ccgattctaa gggccgcttc tttgactccc ggaaggccga cgatgacatg 3600
ccaaagaatg ccgacgccaa cggcgcctat cacatcgccc tgaagggcct gtggtgtctg 3660
gagcagatca gcaagaccga tgacctgaag aaggtgaagc tggccatctc caacaaggag 3720
tggctggagt tcgtgcagac actgaagggc aaaaggccgg cggccacgaa aaaggccggc 3780
caggcaaaaa agaaaaaggg atcctaccca tacgatgttc cagattacgc ttatccctac 3840
gacgtgcctg attatgcata cccatatgat gtccccgact atgcc 3885
Claims (13)
1.一种基于CRISPR/Cas12a的核酸检测系统,其特征在于,包括SsCas12a蛋白和gRNA。
2.根据权利要求1所述的核酸检测系统,其特征在于,所述gRNA的设计原则为:在选取gRNA靶向序列时,靶向序列5’端应具有5’-TTTN-3’序列,且靶向序列本身不形成稳定二级结构、靶向序列和其余序列间不形成稳定二级结构。
3.根据权利要求2所述的核酸检测系统,其特征在于,当靶序列如SEQ ID NO.2所示时,gRNA序列如SEQ ID NO.3所示。
4.根据权利要求1所述的核酸检测系统,其特征在于,SsCas12a蛋白的核苷酸序列如SEQ ID NO.1或SEQ ID NO.6所示。
5.一种基于权利要求1~3任一所述系统的核酸检测方法,其特征在于,是利用SsCas12a蛋白和对应靶标序列的gRNA进行CRISPR核酸检测。
6.SsCas12a蛋白在切割DNA方面的应用。
7.SsCas12a蛋白在作为或制备DNA切割工具方面的应用。
8.SsCas12a蛋白在核酸检测方面的应用。
9.SsCas12a蛋白在基于CRISPR/Cas12a的核酸检测方面的应用。
10.SsCas12a蛋白在作为或制备核酸检测工具方面的应用。
11.SsCas12a蛋白在作为或制备基于CRISPR/Cas12a的核酸检测工具方面的应用。
12.根据权利要求6~11任一所述的应用,其特征在于,SsCas12a蛋白具有靶向体外DNA序列或体内基因组序列进行特异切割的活性。
13.根据权利要求6~11任一所述的应用,其特征在于,SsCas12a蛋白具有gRNA介导的DNA切割活性。
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CN113444777A (zh) * | 2021-07-20 | 2021-09-28 | 安徽医科大学第四附属医院 | 一种用于碳青霉烯酶检测的CrRNA、CRISPR-Cas12a系统及应用 |
CN114410737A (zh) * | 2022-01-24 | 2022-04-29 | 深圳市儿童医院 | 一种可视化检测儿童流感病毒的crispr系统及其方法和应用 |
CN115087750A (zh) * | 2022-03-30 | 2022-09-20 | 中国医学科学院药用植物研究所 | 基于全基因组分析的真核生物物种鉴定方法及应用 |
CN116121446A (zh) * | 2022-08-10 | 2023-05-16 | 厦门大学 | 基于CRISPR-Cas检测猪流感病毒的方法、试剂盒及应用 |
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