CN111778252A - 一种靶向敲除SST基因的sgRNA及其CRISPR/Cas9***和应用 - Google Patents
一种靶向敲除SST基因的sgRNA及其CRISPR/Cas9***和应用 Download PDFInfo
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- CN111778252A CN111778252A CN202010690620.8A CN202010690620A CN111778252A CN 111778252 A CN111778252 A CN 111778252A CN 202010690620 A CN202010690620 A CN 202010690620A CN 111778252 A CN111778252 A CN 111778252A
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Abstract
本发明提供了一种靶向敲除SST基因的sgRNA及其CRISPR/Cas9***和应用,属于基因敲除技术领域。本发明提供了一种靶向敲除SST基因的sgRNA,包括SST sgRNA1和SST sgRNA2,还提供了包含所述sgRNA的CRISPR/Cas9***。将所述CRISPR/Cas9***转染至猪肾成纤维细胞中,筛选得到的阳性细胞,取得较高的基因删除效率,同时CRISPR/Cas9***通过对猪SST基因进行完全性基因敲除,可以提高猪的生长速度,从而提高养猪业的经济效益,同时所述CRISPR/Cas9***对进一步研究SST基因的功能及其与某些疾病的相互关联具有十分重要的意义。
Description
技术领域
本发明属于基因敲除技术领域,具体涉及一种靶向敲除SST基因的sgRNA及其CRISPR/Cas9***和应用。
背景技术
SST基因位于猪13号染色体,长度为1183bp,包含2个外显子。目前普遍认为SST在哺乳动物体内的主要功能是通过直接或间接的作用抑制生长激素的分泌从而调控动物生长。畜牧生产上常采用一些措施降低动物体内SST含量,削弱或消除SST对消化***的抑制,解除SST对生长激素、***1的抑制,进而提高动物生长速度和体型大小。但这些抑制措施不适用于根本性的长期抑制研究。
CRISPR-Cas9基因编辑技术是通过一段短的单链向导RNA(single guide RNA,sgRNA)来识别特定的DNA序列后,引导Cas9蛋白定位到该指定的DNA序列上进行切割形成双链断裂,随后在该双链断裂区进行非同源末端连接或同源重组对损伤的序列进行修复,从而实现对基因组DNA的定向修饰。
目前CRISPR-Cas9***作为基因编辑工具被广泛应用,克服了传统基因编辑技术步骤繁琐、耗时长、效率低等缺点,以其较少的成分、便捷的操作及较高的效率满足了大多数领域的基因编辑需求。然而,现有技术中CRISPR-Cas9***的删除效果有待进一步提高。
发明内容
有鉴于此,本发明的目的在于提供一种靶向敲除SST基因的sgRNA,具有高效特异性结合SST基因。
本发明的目的还在于提供一种靶向敲除SST基因的CRISPR/Cas9***,具有较高的SST基因删除率,可用于猪细胞株的SST基因沉默。
本发明提供了一种靶向敲除SST基因的sgRNA,包括SST sgRNA1和SST sgRNA2;
所述SST sgRNA1的核苷酸序列如序列表SEQ ID No.1所示;
所述SST sgRNA2的核苷酸序列如序列表SEQ ID No.2所示。
本发明提供了一种靶向敲除SST基因的CRISPR/Cas9***,所述CRISPR/Cas9***包括所述sgRNA。
本发明提供了所述CRISPR/Cas9***的构建方法,包括以下步骤:
1)使用BbsI酶切CRISPR/Cas9载体pSpCas9(BB)-2A-Puro,得到线性化后的表达载体;
2)将所述靶向敲除SST基因的SST sgRNA1和SST sgRNA2分别与酶切后的pSpCas9(BB)-2A-Puro载体连接,得到靶向敲除SST基因的pSpCas9(BB)-2A-Puro-SST sgRNA1和pSpCas9(BB)-2A-Puro-SSTsgRNA2,构建得到靶向敲除SST基因的CRISPR/Cas9***。
本发明提供了一种靶向敲除SST基因的试剂盒,包括所述CRISPR/Cas9***。
本发明提供了所述CRISPR/Cas9***或所述试剂盒在制备敲除SST基因的细胞株中的应用。
优选的,所述细胞株包括猪肾成纤维细胞。
优选的,所述SST基因的敲除片段为SEQ ID No.3所示。
优选的,所述敲除SST基因的细胞株的制备方法,将所述靶向敲除SST基因的CRISPR/Cas9***转染至细胞株中,经过阳性筛选,得到敲除SST基因的细胞株。
本发明提供了所述CRISPR/Cas9***或所述试剂盒在畜牧生产中的应用。
本发明提供了一种双sgRNA位点靶向敲除SST基因的鉴定方法,包括以下步骤:
提取待测细胞基因组DNA;
以所述基因组DNA为模板,用扩增引物对进行PCR扩增,所述PCR扩增产物进行检测,野生型片段长度为416bp,基因敲除后片段长度为380bp;所述扩增引物对中,上游引物的核苷酸序列如SEQ ID No.4所示;下游引物的核苷酸序列如SEQ ID No.5所示。
本发明提供了一种靶向敲除SST基因的sgRNA,包括SST sgRNA1和SST sgRNA2。所述SST sgRNA1和SST sgRNA2均以第一外显子为打靶区域,能够快速、高效靶向SST基因,为后续实现SST基因功能失活提供保证。
本发明提供了一种靶向敲除SST基因的CRISPR/Cas9***,所述CRISPR/Cas9***包括所述sgRNA。将所述CRISPR/Cas9***转染至猪肾成纤维细胞中,筛选得到的阳性细胞,共挑选到13个单克隆,检测到发生SST删除突变的阳性细胞为11个,删除效率为78.6%,其中单等位基因删除4个,双等位基因删除7个,双等位基因删除效率为53.8%。
同时本发明提供的CRISPR/Cas9***对猪SST基因进行完全性基因敲除,可以提高猪的生长速度,从而提高养猪业的经济效益,因此可以应用于畜牧养殖中;另一方面,所述CRISPR/Cas9***对进一步研究SST基因的功能及其与某些疾病的相互关联具有十分重要的意义。
附图说明
图1为猪SST-sgRNA1和SST-sgRNA2在猪基因组上的靶向位置示意图;
图2为***有引导序列的Cas9 sgRNA表达载体示意图;
图3-A为pSpCas9(BB)-2A-Puro-SST sgRNA1和pSpCas9(BB)-2A-Puro-SST sgRNA2质粒酶切电泳图;其中,M为DNA marker;1为pSpCas9(BB)-2A-Puro-SST sgRNA1质粒;2为线性化的pSpCas9(BB)-2A-Puro-SST sgRNA1质粒;3为pSpCas9(BB)-2A-Puro-SST sgRNA2质粒;4为线性化的pSpCas9(BB)-2A-Puro-SST sgRNA2;图3-B为pSpCas9(BB)-2A-Puro-SSTsgRNA1和pSpCas9(BB)-2A-Puro-SST sgRNA2质粒测序图;
图4为显微镜观察分离的原代猪肾成纤维细胞的形态图;
图5为SST基因删除示意图;
图6为单克隆细胞PCR产物电泳图。
具体实施方式
本发明提供了一种靶向敲除SST基因的sgRNA,包括SST sgRNA1和SST sgRNA2;所述SST sgRNA1的核苷酸序列如序列表SEQ ID No.1所示;所述SST sgRNA2的核苷酸序列如序列表SEQ ID No.2所示。本发明所述sgRNA是以第一外显子为打靶区域,猪SST sgRNA1和SST sgRNA2这两个位点在猪基因组的准确定位是13号染色体SST编码区1号外显子上(见图1)。设计条件为:①sgRNA的长度为20nt,核心序列为NNNNNNNNNNNNNNNNNNNN(20)-NGG(SEQID No.6);②正义链模板的5’端添加CACCG,反义链模板的5’端添加AAAC,使寡核苷酸链与载体质粒黏性末端互补。编码链模板5’端添加CACC,非编码链模板3’端添加AAAC,与BbsI酶切后形成的粘性末端互补,设计2对CRISPR寡核苷酸链。SST靶向位点及sgRNA寡核苷酸序列依次为SST-sgRNA1oligo编码链(SEQ ID No.7,CACCGGCCGCGCTCTCCATCGTCC)、SST-sgRNA1oligo非编码链(SEQ ID No.8,AAACGGACGATGGAGAGCGCGGCC)、SST-sgRNA2oligo编码链(SEQ ID No.9,CACCGTGACGGAGTCGGGGATCCGA)、SST-sgRNA2oligo非编码链(SEQ IDNo.10,AAACTCGGATCCCCGACTCCGTCAC)。本发明对所述sgRNA的来源没有特殊限制,采用本领域所熟知的sgRNA的来源即可,例如委托基因合成公司合成。
本发明提供了一种靶向敲除SST基因的CRISPR/Cas9***,所述CRISPR/Cas9***包括所述sgRNA。本发明利用sgRNA的靶向性,特异性识别SST基因的特定DNA序列后,引导Cas9蛋白定位到该SST基因的DNA序列上进行切割,形成双链断裂,从而阻止该基因的正常生物学功能,解除SST对生长激素、***1的抑制,进而提高动物生长速度和体型大小。
本发明提供了所述CRISPR/Cas9***的构建方法,包括以下步骤:
1)使用BbsI酶切CRISPR/Cas9载体pSpCas9(BB)-2A-Puro,得到线性化后的表达载体;
2)将所述靶向敲除SST基因的SST sgRNA1和SST sgRNA2分别与酶切后的pSpCas9(BB)-2A-Puro载体连接,得到靶向敲除SST基因的pSpCas9(BB)-2A-Puro-SST sgRNA1和pSpCas9(BB)-2A-Puro-SSTsgRNA2,构建得到靶向敲除SST基因的CRISPR/Cas9***。
在本发明中,所述pSpCas9(BB)-2A-Puro购自Addgene公司。本发明对所述BbsI酶切的方法没有特殊限制,采用本领域所熟知的BbsI酶切方法即可。本发明对所述连接的方法没有特殊限制,采用本领域所熟知的连接方法即可。所述连接后,还优选对所述连接产物进行筛选,经过PCR扩增验证包含目标片段的质粒为阳性质粒。所述pSpCas9(BB)-2A-Puro-SST sgRNA1和pSpCas9(BB)-2A-Puro-SSTsgRNA2的拷贝数比优选为1:1。所述pSpCas9(BB)-2A-Puro-SST sgRNA1和pSpCas9(BB)-2A-Puro-SSTsgRNA2分别以pSpCas9(BB)-2A-Puro载体为骨架,带有hspCas9基因、CRISPRArray和tracrRNA序列的一个表达载体(见图2)。
本发明提供了一种靶向敲除SST基因的试剂盒,包括所述CRISPR/Cas9***。所述试剂盒优选还包括转染用试剂和敲除基因鉴定用试剂。所述转染用试剂包括常见的转染试剂。所述敲除基因鉴定用试剂优选包括PCR扩增引物对、PCR扩增用试剂等。所述PCR扩增引物对的上游引物的核苷酸序列如SEQ ID No.4所示;下游引物的核苷酸序列如SEQ ID No.5所示。在本发明中,所述试剂盒的使用方法,优选包括将所述靶向敲除SST基因的CRISPR/Cas9***转染至细胞株中,经过阳性筛选,得到敲除SST基因的细胞株。优选还包括采用敲除基因鉴定用试剂对所得到的细胞株进行PCR扩增鉴定,野生型片段长度为416bp(SEQ IDNo.11,ACGAGGGTAATGGTGCGTAAAAGCGCTGGTGAGATCTGGGGGCGCCTCCTAGTCTGACGTCAGAGAGAGAGTTTAAAAAGGGGGAGACGGTGGCGAGCGCACAAGCCGCTTCAGGAGTCGCGAGGTTCAGAGCCGTCGCTGCTGCCTGCAAATCGACTCCTAGAGTTTGACCAACCGCGCTCTAGCTCGGCTTCTCTGGCCGCTGCCGAGATGCTGTCCTGCCGCCTCCAGTGCGCGCTGGCCGCGCTCTCCATCGTCCTGGCTCTGGGCGGTGTCACTGGCGCGCCCTCGGATCCCCGACTCCGTCAGTTTCTGCAGAAGTCCCTGGCTGCTGCCGCTGGGAAGCAGGTAAGGAGACTCCCTCGACGCCTTCTTTCCCCTCTCGCGAATCCCCTAACCTTACCTTAGCCTTGCCC,基因敲除后片段长度为380bp(SEQ IDNo.12,ATCCCCGACTCCGTCAGTTTCTGCAGAAGTCCCTGGCTGCTGCCGCTGGGAAGCAGGTAAGGAGACTCCCTCGACGCCTTCTTTCCCCTCTCGCGAATCCCCTAACCTTACCTTAGCCTTGCCCACGAGGGTAATGGTGCGTAAAAGCGCTGGTGAGATCTGGGGGCGCCTCCTAGTCTGACGTCAGAGAGAGAGTTTAAAAAGGGGGAGACGGTGGCGAGCGCACAAGCCGCTTCAGGAGTCGCGAGGTTCAGAGCCGTCGCTGCTGCCTGCAAATCGACTCCTAGAGTTTGACCAACCGCGCTCTAGCTCGGCTTCTCTGGCCGCTGCCGAGATGCTGTCCTGCCGCCTCCAGTGCGCGCTGGCCGCGCTCTCCATCG),根据PCR扩增片段长度判断基因敲除情况。野生型SST基因全序列为1183bp(SEQID No.13),敲除后的SST基因序列为1147bp(SEQ ID No.14)。
本发明提供了所述CRISPR/Cas9***或所述试剂盒在制备敲除SST基因的细胞株中的应用。本发明对所有真核细胞均适用,为了举例说明制备方法,本发明实施例以猪肾成纤维细胞为例详细描述。所述SST基因的敲除片段优选为SEQ ID No.3所示(TCCTGGCTCTGGGCGGTGTCACTGGCGCGCCCTCGG)。所述敲除SST基因的细胞株的制备方法,优选将所述靶向敲除SST基因的CRISPR/Cas9***转染至细胞株中,经过阳性筛选,得到敲除SST基因的细胞株。
本发明提供了所述CRISPR/Cas9***或所述试剂盒在畜牧生产中的应用。所述畜牧生产中,所述CRISPR/Cas9***或所述试剂盒通过靶向SST基因,阻断该基因的正常生物学功能,解除SST对生长激素、***1的抑制,对提高动物生长速度和体型大小有帮助。
本发明提供了一种双sgRNA位点靶向敲除SST基因的鉴定方法,包括以下步骤:提取待测细胞基因组DNA;
以所述基因组DNA为模板,用扩增引物对进行PCR扩增,所述PCR扩增产物进行检测,野生型片段长度为416bp,基因敲除后片段长度为380bp;所述扩增引物对中,上游引物的核苷酸序列如SEQ ID No.4所示;下游引物的核苷酸序列如SEQ ID No.5所示。
本发明对述提取待测细胞基因组DNA的方法没有特殊限制,采用本领域所熟知的提取方法即可,例如试剂盒提取。
在本发明中,所述PCR扩增体系为:Premix Taq 10μL,SST-E1-F 1μL,SST-E1-R 1μL,DNA模板2μL,ddH2O 6μL;所述PCR扩增反应程序为94℃预变形2min,94℃变性30s,57℃退火30s,72℃延伸30s,30个循环,72℃总延伸5min。采用双sgRNA作为靶向RNA,与单独使用一种sgRNA相比,具有更高效的靶向沉默SST基因的作用,取得了较高的基因删除效率。
下面结合实施例对本发明提供的一种靶向敲除SST基因的sgRNA及其CRISPR/Cas9***和应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
构建Cas9 sgRNA敲除质粒
1.1 sgRNA的筛选及合成
选定猪SST基因(GenBank登陆号:NC_010455.5)第一外显子为打靶区域,利用网站(http://crispor.tefor.net)对打靶区域设计sgRNA,设计条件为:①sgRNA的长度为20nt,核心序列为NNNNNNNNNNNNNNNNNNNN(20)-NGG(SEQ ID No.6);②正义链模板的5’端添加CACCG,反义链模板的5’端添加AAAC,使寡核苷酸链与载体质粒黏性末端互补。最终选择两个合适的靶点,命名为SST-sgRNA1和SST-sgRNA2。编码链模板5’端添加CACC,非编码链模板3’端添加AAAC,与BbsI酶切后形成的粘性末端互补,设计2对CRISPR寡核苷酸链。
SST靶向位点及sgRNA寡核苷酸序列:
实施例2
载体构建
1.2.1使用BbsI酶切pSpCas9(BB)-2A-Puro质粒,酶切反应体系如下:
成分 | 添加量 |
pSpCas9(BB)-2A-Puro质粒 | 1μg |
BbsI酶 | 2μL |
FDbuffer | 2μL |
ddH<sub>2</sub>O | 至50μL |
Total | 50μL |
在冰上操作,将以上成分依次加入,充分混匀后,37℃循环水浴中酶切2h。
1.2.2按照诺唯赞凝胶回收试剂盒说明书回收上述酶切产物。
1.2.3 sgRNA oligo退火及双链的形成
将公司合成的每对寡核苷酸稀释至10μmol/L,按以下比例混合:16μL ddH2O,2μL10×NEB Buffer 3,1μL正义链,1μL反义链,混匀后95℃变性5min,之后在室温下自然退火1h形成双链。
1.2.4 pSpCas9(BB)-2A-Puro-SST sgRNA质粒构建
将寡核苷酸双链SST sgRNA1和SST sgRNA2分别与线性化的pSpCas9(BB)-2A-Puro连接,连接反应体系为:1μL线性化的PX459,3μL退火产物,5μL SolutionⅠ,加ddH2O至10μL。将以上成分充分混合均匀后放在37℃水浴连接2h;连接完成后置于冰上用于转化。
1.2.5转化大肠杆菌DH5α
将5μL连接产物加入到50μL感受态细胞DH5α中,轻弹混匀,冰置30min,42℃热激90s,取出后再冰置5min,先加入1mL无抗体的培养基摇配复苏45min,再涂布于含氨苄抗体的平板上,挑取单克隆继续培养,提取质粒后酶切验证,并通过测序检测是否连接正确,测序引物为通用引物U6。图3为质粒酶切和测序结果。由图3可知,成功构建重组质粒,分别命名为pSpCas9(BB)-2A-Puro-SST sgRNA1和pSpCas9(BB)-2A-Puro-SST sgRNA2。
实施例3
电穿孔法转染猪肾成纤维细胞
1.1主要试剂与材料
1-3日龄雄性大白仔猪来自湖北省农业科学院畜牧兽医研究所试验猪场;G418抗生素购自Sigma公司;DMEM、DPBS、胎牛血清、DMSO等细胞培养和冻存试剂购自Gibco公司。电转缓冲液由实验室自行配制。
1.2猪肾成纤维细胞分离培养
取1头新生1~3d的雄性大白猪,经腹腔注射戊巴比妥钠(50mg)深度麻醉;用75%酒精充分清洗消毒大白猪体表,然后用灭菌医用手术器械无菌操作取出双侧肾脏,置于75%酒精中浸泡并移至超净工作台内。
用含2%抗生素(青霉素132mg/L,链霉素200mg/L)的DPBS反复清洗肾脏,然后用手术剪仔细剥离肾脏包膜,再用75%酒精浸泡大约30s以充分消毒避免污染。用DPBS清洗2次后将肾脏组织放入60mm直径细胞培养皿中,用眼科剪将其剪成大小约为1mm3的小块组织。
将小块组织分散到六孔板中,每孔约9块组织,并尽量吸干组织周围的液体。在恒温培养箱中倒置4h,待组织块与培养皿底部紧密粘连,加入500μL培养基,培养基中含15%血清,1%双抗。在39℃,5%CO2细胞培养温箱中培养24h后,加入2mL完全培养基。每天观察细胞的形态和生长状态,根据细胞的状态进行换液,预防细胞受到污染。经过7d左右的培养,成纤维细胞在组织块周围长成片并汇合度逐渐达到90%时,将组织块挑去可获得原代细胞。肾成纤维细胞生长状态较好,呈梭形,显微镜下观察细胞透明,边缘清晰。传代后生长迅速,约2~3d既可长满六孔板。转染前一天消化细胞传代后,转染时细胞即处于对数生长期,活力最好,转染效果最佳(见图4)。
1.3猪肾成纤维细胞的培养与电穿孔转染
用含15%胎牛血清、1%双抗的DMEM培养基,在CO2浓度为5%,温度为37℃的培养箱中培养成纤维细胞。转染前一天,将猪肾成纤维细胞用胰酶消化为单细胞悬液后,按照0.25~1×106细胞/孔将细胞接种于6孔板中,待细胞处于对数生长期、汇合度为80%左右时进行电穿孔转染两个重组质粒共同转染,没有将两个质粒分开进行转染的实验。转染条件为120V/cm,脉冲时间3ms,质粒添加量4mg/mL。
1.4嘌呤霉素筛选
细胞转染48h后,重新接种细胞,使六孔板每个孔中约有10000个细胞,继续培养,细胞恢复到较好的状态。继续培养48h后,换新的培养基,并加入嘌呤霉素,嘌呤霉素的浓度为1.75ng/μL,每天观察细胞的存活状态。嘌呤霉素加入72h左右,换新的不含嘌呤霉素的培养基,提取细胞的DNA,进行PCR检测。
1.5单克隆细胞的筛选及培养
待经过嘌呤霉素筛选后的单个细胞增殖为细胞团后,在显微镜下观察大小适度的细胞团,并用记号笔在培养皿的底部标记细胞团的位置。移除培养基,用DPBS清洗两遍。用无菌镊子夹起一个克隆环,将克隆环底部粘上无菌凡士林,轻轻地将克隆环放在一个选定的克隆之上,用镊子稍用力均匀按压以克隆环能稳定立在培养皿且密封上为止。加50μL0.25%的胰酶到克隆环里,细胞开始变圆时加100μL的培养基,吹起细胞并吸取到96孔板中,再加100μL的培养基到克隆环,吹吸后加到对应的孔中。2d换一次培养基,待96孔板中的细胞长满传代至24孔板,最后传代至6孔板中。
实施例4
单克隆细胞的基因型鉴定
分别提取未转染的细胞、转染后48h的细胞、上述制备的单克隆扩繁的细胞的DNA进行PCR扩增。引物在第一外显子两侧设计,序列分别为SST-E1-F:5’-ACGAGGGTAATGGTGCGTAA-3’(SEQ ID No.4),SST-E1-R:5’-CCTTACCTTAGCCTTGCCC-3’(SEQID No.5)野生型片段416bp,编辑后片段380bp。PCR扩增体系为:Premix Taq 10μL,SST-E1-F 1μL,SST-E1-R 1μL,DNA模板2μL,ddH2O 6μL,94℃预变形2min;反应程序为:94℃变性30s,57℃退火30s,72℃延伸30s,30个循环,72℃总延伸5min。PCR扩增产物用90V的电压,2.5%的琼脂糖凝胶电泳分离,并将单克隆细胞的PCR产物送至公司测序。
基因删除位置见图5。测序结果为沉默后的SST基因的核苷酸序列如SEQ ID No.12所示,而野生型SST基因的核苷酸序列如SEQ ID No.11所示。
统计基因删除效率
从上述实验中一共挑选到13个单克隆,PCR产物电泳结果见6。在sgRNA1和sgRNA2靶点两侧设计引物,扩增两个靶点之间的片段。1号泳道为100bp DNA Ladder,2-14号泳道为筛选到的单克隆细胞模板扩增结果。其中两个克隆(2和3号泳道)没有片段缺失,四个克隆(4/5/6/7号泳道)为单等位基因缺失,七个克隆(8/9/10/11/12/13/14号泳道)双等位基因片段缺失。结果表明,检测到发生SST删除突变的阳性细胞为11个,删除效率为78.6%(11/13),其中单等位基因删除4个,双等位基因删除7个(单等位基因删除表示一对同源染色体的其中一个染色体上的基因删除,另一个等位基因没有删除;双等位基因删除表示一对同源染色体上控制SST表达的两个等位基因都删除了),双等位基因删除效率为53.8%(7/13)。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 湖北省农业科学院畜牧兽医研究所
<120> 一种靶向敲除SST基因的sgRNA及其CRISPR/Cas9***和应用
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tgacggagtc ggggatccga 20
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gccgcgctct ccatcgtcc 19
<210> 3
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tcctggctct gggcggtgtc actggcgcgc cctcgg 36
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
acgagggtaa tggtgcgtaa 20
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ccttacctta gccttgccc 19
<210> 6
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
nnnnnnnnnn nnnnnnnnnn ngg 23
<210> 7
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
caccggccgc gctctccatc gtcc 24
<210> 8
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aaacggacga tggagagcgc ggcc 24
<210> 9
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
caccgtgacg gagtcgggga tccga 25
<210> 10
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aaactcggat ccccgactcc gtcac 25
<210> 11
<211> 416
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
acgagggtaa tggtgcgtaa aagcgctggt gagatctggg ggcgcctcct agtctgacgt 60
cagagagaga gtttaaaaag ggggagacgg tggcgagcgc acaagccgct tcaggagtcg 120
cgaggttcag agccgtcgct gctgcctgca aatcgactcc tagagtttga ccaaccgcgc 180
tctagctcgg cttctctggc cgctgccgag atgctgtcct gccgcctcca gtgcgcgctg 240
gccgcgctct ccatcgtcct ggctctgggc ggtgtcactg gcgcgccctc ggatccccga 300
ctccgtcagt ttctgcagaa gtccctggct gctgccgctg ggaagcaggt aaggagactc 360
cctcgacgcc ttctttcccc tctcgcgaat cccctaacct taccttagcc ttgccc 416
<210> 12
<211> 380
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atccccgact ccgtcagttt ctgcagaagt ccctggctgc tgccgctggg aagcaggtaa 60
ggagactccc tcgacgcctt ctttcccctc tcgcgaatcc cctaacctta ccttagcctt 120
gcccacgagg gtaatggtgc gtaaaagcgc tggtgagatc tgggggcgcc tcctagtctg 180
acgtcagaga gagagtttaa aaagggggag acggtggcga gcgcacaagc cgcttcagga 240
gtcgcgaggt tcagagccgt cgctgctgcc tgcaaatcga ctcctagagt ttgaccaacc 300
gcgctctagc tcggcttctc tggccgctgc cgagatgctg tcctgccgcc tccagtgcgc 360
gctggccgcg ctctccatcg 380
<210> 13
<211> 1183
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atgctgtcct gccgcctcca gtgcgcgctg gccgcgctct ccatcgtcct ggctctgggc 60
ggtgtcactg gcgcgccctc ggatccccga ctccgtcagt ttctgcagaa gtccctggct 120
gctgccgctg ggaagcaggt aaggagactc cctcgacgcc ttctttcccc tctcgcgaat 180
cccctaacct taccttagcc ttgcccctcc tcccttgggt ggacttagga ggtggtccca 240
aagagtatcg gtgcttttct gggtccctta ggcaccaaat ctctcaggaa aactttcaaa 300
gtccagaatt cctttttacc tctttgtttt ttccctcttt gatcagcgca gtaggtcaca 360
gttcaggtga gttctttggc tttcaagaaa attctaagat ctggggaact gagctcgagg 420
ggatgatggc atctatccgc ggtgctgacc atgggaggtg ctgacccagg tgctgaaagc 480
gcggacctct gaagcttcct aagcagtacc tcccacccat gcagcagggc tgggggctga 540
agggcacaac agctagaaca caatatgttt acgactgtga aaagtcttgt ttcccacagt 600
tgatttagta aaaatggtaa gaacaattct attttgtagc tcatgatatg gaaattgagt 660
taaataaaat gttggcatat attttacctt tgctaactaa tgatgttcta tttctttcta 720
tgtgtgagtt ctaaacctgt agacactaaa accttgcaga aacatttgag tttttaaagc 780
ttccttgtgt aacttggtaa attataacaa cagccctgtt tttatatcct taacctattt 840
taagcattac acaaagtgca catagaaaat tgggggttgg atgtgattaa atctgttttt 900
taaaccaagc ttttccattt ctttttcact atcctcattc tcatccccct ccccctccca 960
ttccacacag gaactggcca agtacttctt ggcggagctg ctctctgaac ccaaccagac 1020
agagaacgat gccctggagc ctgaagattt gtcccaggct gctgagcagg atgaaatgag 1080
gctggagctg cagagatcag ctaactcaaa cccggccatg gcaccccgag aacgcaaagc 1140
tggctgcaag aatttcttct ggaagacttt cacatcctgt tag 1183
<210> 14
<211> 1147
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgctgtcct gccgcctcca gtgcgcgctg gccgcgctct ccatcgatcc ccgactccgt 60
cagtttctgc agaagtccct ggctgctgcc gctgggaagc aggtaaggag actccctcga 120
cgccttcttt cccctctcgc gaatccccta accttacctt agccttgccc ctcctccctt 180
gggtggactt aggaggtggt cccaaagagt atcggtgctt ttctgggtcc cttaggcacc 240
aaatctctca ggaaaacttt caaagtccag aattcctttt tacctctttg ttttttccct 300
ctttgatcag cgcagtaggt cacagttcag gtgagttctt tggctttcaa gaaaattcta 360
agatctgggg aactgagctc gaggggatga tggcatctat ccgcggtgct gaccatggga 420
ggtgctgacc caggtgctga aagcgcggac ctctgaagct tcctaagcag tacctcccac 480
ccatgcagca gggctggggg ctgaagggca caacagctag aacacaatat gtttacgact 540
gtgaaaagtc ttgtttccca cagttgattt agtaaaaatg gtaagaacaa ttctattttg 600
tagctcatga tatggaaatt gagttaaata aaatgttggc atatatttta cctttgctaa 660
ctaatgatgt tctatttctt tctatgtgtg agttctaaac ctgtagacac taaaaccttg 720
cagaaacatt tgagttttta aagcttcctt gtgtaacttg gtaaattata acaacagccc 780
tgtttttata tccttaacct attttaagca ttacacaaag tgcacataga aaattggggg 840
ttggatgtga ttaaatctgt tttttaaacc aagcttttcc atttcttttt cactatcctc 900
attctcatcc ccctccccct cccattccac acaggaactg gccaagtact tcttggcgga 960
gctgctctct gaacccaacc agacagagaa cgatgccctg gagcctgaag atttgtccca 1020
ggctgctgag caggatgaaa tgaggctgga gctgcagaga tcagctaact caaacccggc 1080
catggcaccc cgagaacgca aagctggctg caagaatttc ttctggaaga ctttcacatc 1140
ctgttag 1147
Claims (10)
1.一种靶向敲除SST基因的sgRNA,其特征在于,包括SST sgRNA1和SST sgRNA2;
所述SST sgRNA1的核苷酸序列如序列表SEQ ID No.1所示;
所述SST sgRNA2的核苷酸序列如序列表SEQ ID No.2所示。
2.一种靶向敲除SST基因的CRISPR/Cas9***,其特征在于,所述CRISPR/Cas9***包括权利要求1所述sgRNA。
3.权利要求2所述CRISPR/Cas9***的构建方法,其特征在于,包括以下步骤:
1)使用BbsI酶切CRISPR/Cas9载体pSpCas9(BB)-2A-Puro,得到线性化后的表达载体;
2)将所述靶向敲除SST基因的SST sgRNA1和SST sgRNA2分别与酶切后的pSpCas9(BB)-2A-Puro载体连接,得到靶向敲除SST基因的pSpCas9(BB)-2A-Puro-SST sgRNA1和pSpCas9(BB)-2A-Puro-SSTsgRNA2,构建得到靶向敲除SST基因的CRISPR/Cas9***。
4.一种靶向敲除SST基因的试剂盒,其特征在于,包括权利要求2所述CRISPR/Cas9***。
5.权利要求2所述CRISPR/Cas9***或权利要求4所述试剂盒在制备敲除SST基因的细胞株中的应用。
6.根据权利要求5所述应用,其特征在于,所述细胞株包括猪肾成纤维细胞。
7.根据权利要求5所述应用,其特征在于,所述SST基因的敲除片段为SEQ ID No.3所示。
8.根据权利要求5~7任意一项所述应用,其特征在于,所述敲除SST基因的细胞株的制备方法,将权利要求2所述靶向敲除SST基因的CRISPR/Cas9***转染至细胞株中,经过阳性筛选,得到敲除SST基因的细胞株。
9.权利要求2所述CRISPR/Cas9***或权利要求4所述试剂盒在畜牧生产中的应用。
10.一种双sgRNA位点靶向敲除SST基因的鉴定方法,其特征在于,包括以下步骤:
提取待测细胞基因组DNA;
以所述基因组DNA为模板,用扩增引物对进行PCR扩增,所得PCR扩增产物进行检测,根据野生型片段长度为416bp,基因敲除后片段长度为380bp判断基因敲除情况;在所述扩增引物对中,上游引物的核苷酸序列如SEQ ID No.4所示;下游引物的核苷酸序列如SEQ IDNo.5所示。
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CN114606199B (zh) * | 2022-03-21 | 2023-10-31 | 上海科技大学 | 一种目标基因片段缺失突变体细胞的制备方法 |
CN114736972A (zh) * | 2022-05-12 | 2022-07-12 | 岭南师范学院 | 一种用于评估四指马鲅生长相关性状的试剂 |
CN114736972B (zh) * | 2022-05-12 | 2024-01-30 | 岭南师范学院 | 一种用于评估四指马鲅生长相关性状的试剂 |
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