CN108018288A - 用于突变rosa26基因的系统及其应用 - Google Patents
用于突变rosa26基因的系统及其应用 Download PDFInfo
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- CN108018288A CN108018288A CN201810038608.1A CN201810038608A CN108018288A CN 108018288 A CN108018288 A CN 108018288A CN 201810038608 A CN201810038608 A CN 201810038608A CN 108018288 A CN108018288 A CN 108018288A
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Abstract
本发明公开了用于突变ROSA26基因的系统及其应用。本发明提供的用于突变ROSA26基因系统为CRISPR/Cas9系统，该系统包括靶向ROSA26基因的sgRNA，sgRNA的靶序列为如下A1)、A2)或A3)：A1)序列表中序列2的核苷酸序列；A2)与A1)限定的DNA序列具有75％或75％以上同一性的由A1)衍生的DNA序列；A3)在严格条件下与A1)限定的DNA序列杂交的由A1)衍生的DNA序列；该系统还包括Cas9和含有Sfat1基因表达盒的供体DNA。实验证明，利用本发明的系统可以成功敲除ROSA26基因，并定点整合Sfat1基因，最终成功得到表达Sfat1的动物。
Description
技术领域
本发明涉及生物技术领域中，用于突变ROSA26基因的系统及其应用。
背景技术
多不饱和脂肪酸(PUFAs)是一类被广泛研究和关注的脂肪酸。PUFAs主要包括ω-6和ω-3PUFAs两种类型，每一种类型都包括多个从短碳链(18C)到长碳链(22C)的、且含有多个不饱和键的脂肪酸。越来越多的研究证明，PUFAs具有广泛的生物学功能，它们参与细胞膜的构成并作为许多细胞应答过程中的信号分子，与人类的多种疾病的发生紧密相关，其适宜的含量对于人类及其他哺乳动物的正常发育和保持良好的健康状况极其重要。ω-6PUFAs从总体上来说对人体意义不大，其过量的摄入反而危害人体健康。ω-3PUFAs已经被证实在预防和治疗心血管疾病、关节炎、癌症等多种疾病方面有着重要作用。其中特别是22碳六烯的DHA和20碳五烯的EPA，对人体健康具有重要的积极意义。
通过转基因技术在猪肉中生产ω-3PUFAs是目前优质猪育种的一个热点和方向。ω-3脂肪酸去饱和酶基因是ω-3PUFAs合成的关键基因，它们利用ω-6PUFAs为底物在脂肪酸碳链甲基端第三个碳催化生成一个不饱和键(烯键)而合成相应的ω-3PUFAs。近年来，各国的科学家克隆了多个ω-3脂肪酸去饱和酶基因，但其中大部分基因只能合成18碳的ω-3PUFAs，开发利用价值不大。虽然也有报道能够合成更长链的ω-3PUFAs基因，如真菌Saprolegnia diclina的ω-3脂肪酸去饱和酶能够合成20碳的ω-3PUFAs，另一种真菌Mortierella alpina的1S-4不但能够合成20碳的ω-3PUFAs，还能合成18碳的ω-3PUFAs。真正被用于此类研究的基因是来自于线虫C.elegans的ω-3脂肪酸去饱和酶基因(Fat1)。该基因被转入哺乳动物细胞以及小鼠中能使从18碳到22碳的ω-3PUFAs的含量增加，显示出来很大的可开发利用价值。来自于线虫C.briggasae的ω-3脂肪酸去饱和酶基因Sfat1(similar to Fat1)，可打通ω-6向ω-3多不饱和脂肪酸转化的途径，制备的随机整合Sfat1转基因克隆猪，ω-3脂肪酸的含量有大幅度提高，Sfat1转基因猪肌肉组织中20碳的EPA约占总脂肪酸含量的16％，22碳的DHA约占总脂肪酸含量的4％。转入Sfat1基因构建转基因克隆猪是制备富含ω-3多不饱和脂肪酸转基因猪的有效手段。
迄今为止猪胚胎干细胞尚未成功建系，利用诱导多能干细胞技术制备的猪诱导多能干细胞并不具有生殖系嵌合能力，制备基因修饰猪主要依赖于体细胞基因修饰和体细胞核移植技术。目前，体细胞的基因修饰主要有宿主基因定点整合和外源基因随机插入两种方法。利用常规载体、转座子载体或病毒载体将目的基因整合到宿主基因组中，其整合过程属随机过程，目的基因整合在宿主基因组中的位置与拷贝数不可控。利用随机转基因主要存在以下问题：(1)外源基因整合宿主基因组位置不可控，有可能对宿主基因造成破坏，尤其是使用转座子载体和病毒载体时其更偏向于整合到宿主转录活性区；(2)外源基因在宿主基因组中的拷贝数不确定，所获得的基因修饰动物在传代过程中基因型不一致；(3)外源基因的随机整合导致其在宿主各个组织以及个体之间表达差异及个体表型不一；(4)外源基因的随机插入，尤其是利用病毒载体时其外源基因启动子容易被甲基化，造成外源基因沉默。这些缺点严重制约了基因修饰猪的培育与应用。
CRISPR/Cas9起源于一种高效的细菌免疫防御机制，是很多细菌和大部分古生菌的天然免疫系统，通过对入侵的病毒和核酸进行特异性的识别，利用Cas9蛋白进行切割，从而达到对自身的保护。基于此系统开发的CRISPR/Cas9基因编辑技术，可以方便、高效地实现对基因组的定点剪切，实现对目的基因的快速编辑。有效解决了常规转基因修饰造成的诸多问题，为制备外源基因表达稳定、表型一致的模型提供了有效的途径，大大推动基因修饰大动物模型的研究与应用。基因定点修饰时，需要为一些外源基因，如外源性优良性状基因提供一个安全可靠的停泊“港口”，在这些“港口”外源基因既可稳定高效表达，又对细胞和组织无已知的副作用。转基因小鼠的研究表明：ROSA26基因位点在发育的各个时期，几乎所有的细胞都能高效的介导外源基因的表达，且ROSA26基因的缺失对细胞的活力和功能不产生影响。
CRISPR/Cas9系统是细菌和古细菌中抵御外来入侵微生物的一种防御机制，后被改造成为基因编辑系统，与TALEN(transcription activator-like effector nuclease)以及ZFN(zinc-finger nuclease)技术并列成为三大基因组编辑工具。CRISPR-Cas9系统由三部分元件构成，Cas9核酸内切酶、crRNA和tracrRNA，crRNA可特异识别某一段DNA序列，tracrRNA起连接crRNA和Cas9核酸内切酶的作用，Cas9核酸内切酶在crRNA-tracrRNA的引导下可定点切割DNA，造成DNA双链断裂，细胞启动自我修复机制，通过非末端同源链接进行修复，并产生移码突变(插入或者缺失)而造成基因功能的丧失，实现基因敲除的目的。crRNA与tracrRNA还可以被“改装”成一个向导RNA(single-guide RNA,sgRNA)，这个sgRNA足以帮助Cas9核酸内切酶对DNA进行定点切割，实现基因敲除。
发明内容
本发明的目的是提供用于突变ROSA26基因的CRISPR/Cas9系统，该系统还可以制备富含不饱和脂肪酸动物。
本发明提供的用于突变ROSA26基因的CRISPR/Cas9系统，将该系统记为系统1，所述系统1包括靶向ROSA26基因的sgRNA，所述sgRNA的靶序列为如下A1)、A2)或A3)：
A1)序列表中序列2的核苷酸序列；
A2)与A1)限定的DNA序列具有75％或75％以上同一性的由A1)衍生的DNA序列；
A3)在严格条件下与A1)限定的DNA序列杂交的由A1)衍生的DNA序列。
所述系统1还可包括Cas9。
所述系统1还可包括供体DNA，所述供体DNA含有用于同源重组的ROSA26基因中位于所述靶序列上下游的两个DNA片段。
所述供体DNA还含有Sfat1编码基因或Sfat1编码基因的表达盒。
所述Sfat1的序列可为序列表SEQ ID NO.10所示的蛋白质。
所述Sfat1编码基因具体可为序列表中SEQ ID NO.9的第2744-3943位所示的DNA分子。
所述表达盒具体可为序列表中SEQ ID NO.9的第1400-4442位。
所述供体DNA还可含有neo基因。在本发明的一个实施例中，所述供体DNA为序列表中SEQ ID NO.9所示的DNA片段，其中，SEQ ID NO.9的第1-8位为Not I的识别序列，第9-998位为左同源臂序列，第998-1003位为Hpa I的识别序列，第1400-2641位为CAG启动子的序列，第2744-3943位为Sfat1的基因序列，第4030-4442位为兔β-globulin polyA的序列，第4504-4538位为loxP的序列，第4564-5070位为PGK启动子序列，第5081-5884位为neo基因序列，第6375-6408位为loxP的序列，第6414-6419位为Sal I识别序列，第6420-7970位为右同源臂序列，第7987-7970为Not I的识别序列。
所述系统1可仅由所述sgRNA和Cas9组成，还可由所述sgRNA、Cas9和所述供体DNA组成。
本发明还提供了用于突变ROSA26基因的CRISPR/Cas9系统，将该系统记为系统2，所述系统2包括与所述sgRNA相关的生物材料，所述生物材料为如下B1)-B8)中的任一种：
B1)编码所述sgRNA的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体；
B4)含有B2)所述表达盒的重组载体；
B5)含有B1)所述核酸分子的细胞系；
B6)含有B2)所述表达盒的细胞系；
B7)含有B3)所述重组载体的细胞系；
B8)含有B4)所述重组载体的细胞系。
所述系统2还包括与Cas9相关的生物材料，所述生物材料为如下C1)-C8)中的任一种：
C1)编码Cas9的核酸分子；
C2)含有C1)所述核酸分子的表达盒；
C3)含有C1)所述核酸分子的重组载体；
C4)含有C2)所述表达盒的重组载体；
C5)含有C1)所述核酸分子的细胞系；
C6)含有C2)所述表达盒的细胞系；
C7)含有C3)所述重组载体的细胞系；
C8)含有C4)所述重组载体的细胞系。
所述系统2还可包括所述供体DNA。
所述系统2可由与所述sgRNA相关的生物材料以及与Cas9相关的生物材料组成，还可由与所述sgRNA相关的生物材料、与Cas9相关的生物材料以及所述供体DNA组成。
本发明还提供了所述系统1、所述系统2或所述sgRNA的靶序列的下述任一应用：
Y1)在突变ROSA26基因中的应用；
Y2)在制备ROSA26基因突变动物细胞中的应用；
Y3)在制备ROSA26基因突变动物中的应用；
Y4)在制备表达Sfat1动物细胞中的应用；
Y5)在制备表达Sfat1动物中的应用；
Y6)在制备富含不饱和脂肪酸动物中的应用；
Y7)在动物育种中的应用。
所述动物可为哺乳动物。所述哺乳动物可为猪。所述细胞可为动物细胞，如猪细胞。进一步，所述细胞可为猪成纤维细胞，如猪胎儿成纤维细胞。
实验证明，利用本发明的系统可以成功敲除ROSA26基因，并定点整合Sfat1基因，最终成功得到表达Sfat1的动物。本发明将转基因育种技术与常规育种技术有机结合，是加速优质转基因猪新品种培育的重要手段和有效途径，将大大加快猪种质资源创新和新品种培育的速度和效率。
附图说明
图1为荧光素酶活性法测定不同sgRNA的Cas9剪切效率。其中，sgRNA1-8分别表示转染CRISPR/Cas9-sgRNA1-8的反应体系，Control表示试剂盒提供的阳性对照。
图2为Surveyor酶切法测定不同sgRNA的Cas9剪切效率。其中，Marker表示DNA分子量标准，Control1和Control2分别表示试剂盒提供的两个对照sgRNA，sgRNA2、3、6和8分别表示转染CRISPR/Cas9-sgRNA2、3、6和8的反应体系。
图3为pLoxP-Sfat1的结构示意图。
图4为利用不同的限制性内切酶对pLoxP-Sfat1进行酶切鉴定结果。其中，Matker为DNA分子量标准。
图5为ROSA26位点定点整合Sfat1基因示意图及单细胞克隆鉴定图。其中，LA为左同源臂，PA为兔β-globulin polyA，RA为右同源臂；Cre+表示经过Cre腺病毒处理，Cre-表示未经过Cre腺病毒处理；M为DNA分子量标准，各电泳图中从上到下的条带大小依次为4500、3000、2000、1200、800、500和200bp。
图6为ROSA26位点定点整合转Sfat1基因猪的鉴定图。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应DNA的5′末端核苷酸，末位均为相应DNA的3′末端核苷酸。
Lipofectamine 2000转染试剂，INVITROGEN公司,REF.11668-030,LOT.1828123；
DEME培养基，GIBCO公司，REF.C11995500CP，LOT.8116413。
实施例1、采用CRISPR/Cas9技术敲除ROSA26基因
一、设计靶向ROSA26基因的sgRNA的靶序列
根据GenBank公布的猪ROSA26基因序列，设计CRISPR/Cas9技术中靶向ROSA26基因的八个sgRNA(记为sgRNA1-8)的靶DNA序列，八个sgRNA的靶DNA序列如表1。
表1 sgRNA的靶DNA序列
二、构建靶向ROSA26基因的CRISPR/Cas9-sgRNA载体
分别在步骤一的八个sgRNA的靶DNA的正向序列5’端加入CACC接头序列，在反向互补序列5’端加入AAAC接头序列，合成sgRNA的靶DNA的正向和反向互补序列，其序列见表2。
将每条序列用ddH2O溶解至100μM，分别取每个sgRNA的靶DNA的两条序列溶液15μl混合，置于95℃变性5min，而后以0.2℃/s的速度匀速降温至16℃，得到八个sgRNA的靶DNA。
表2组建CRISPR/Cas9-sgRNA载体用各sgRNA靶DNA的寡核苷酸序列
用NEB公司的BsmB I酶切CRISPR/Cas9载体(百奥赛图基因生物技术有限公司的UCA CRISPR/Cas9快速构建及活性检测试剂盒中载体)(酶切体系为30μl，其中含有CRISPR/Cas9载体2μg，BsmB I内切酶2μl，1×NEB Buffer 3.1)，55℃孵育2h，孵育完毕后使用1％的琼脂糖凝胶检测酶切结果，使用DNA凝胶回收试剂盒回收载体骨架大片段，NanoDrop测定其浓度在20μg/μl，使用T4DNA连接酶将载体骨架大片段分别与各个sgRNA的靶DNA连接，将得到的序列正确的重组的CRISPR/Cas9-sgRNA载体分别命名为CRISPR/Cas9-sgRNA1、CRISPR/Cas9-sgRNA2、CRISPR/Cas9-sgRNA3、CRISPR/Cas9-sgRNA4、CRISPR/Cas9-sgRNA5、CRISPR/Cas9-sgRNA6、CRISPR/Cas9-sgRNA7和CRISPR/Cas9-sgRNA8，这八个重组载体分别含有sgRNA1-8的靶DNA，分别能表达靶向这八个靶DNA的sgRNA以及Cas9。
三、猪胎儿成纤维细胞的制备
选择妊娠35天的母猪，剖腹手术取出胚胎，放入含双抗的D-PBS中洗涤3次，洗至无色。将洗涤好的胚胎转移到另一平皿中,用无菌剪子剪掉头、四肢和尾,去掉内脏，继续用D-PBS缓冲液洗涤数次，洗至无色，将剩余的部分放入到100mm的平皿中，然后用剪子将胚胎剪切成2～3mm小块，将碎块转移到50ml离心管中。加入二分之一离心管体积的0.25％Trypsin-0.04％EDTA消化液，室温消化15～20min左右，中间吹打5次。加入等体积的终止液10％胎牛血清的DMEM培养液，并轻轻吹打。800rpm/min离心5min，弃上清液，然后加入5ml的含20％胎牛血清的DMEM培养液，轻轻吹打重新悬浮细胞，进行细胞计数。以2×105个细胞/ml的浓度接种于T25细胞培养瓶中，每48h更换一次全培养液，待细胞生长至80％汇合时，将细胞冻存或传代培养，即得到猪胎儿成纤维细胞。
四、荧光素酶活性法测定不同sgRNA的Cas9的剪切效率
复苏猪胎儿成纤维细胞，使用含有10％FBS的DMEM培养基培养，次日待细胞达到90％汇合度后，弃培养上清，PBS清洗细胞一次，后提取细胞基因组DNA，使用引物对1(CCTGAAGGACGAGACTAGCTCTAC和TCTGACCGTAAGGATGCAAGTGAG)扩增sgRNA靶向的猪ROSA26基因片段，并对得到的PCR产物进行测序，将所得测序序列与Genbank公布的Rosa26基因序列比对，检测sgRNA的靶DNA序列在该猪胎儿成纤维细胞中有无突变，结果显示该猪胎儿成纤维细胞中sgRNA1-8的靶DNA无突变。
传代293T细胞(ATCC)(所用细胞培养基为含10％FBS的DEME培养基)至96孔板(10000细胞/孔)，次日用脂质体转染法转染上述8个CRISPR/Cas9-sgRNA载体和precutpUCA(LUC)-ROSA26-1载体(百奥赛图基因生物技术有限公司的UCA CRISPR/Cas9快速构建及活性检测试剂盒中载体)，其中每个转染体系中含有Lipofectamine 2000转染试剂2μl，CRISPR/Cas9-sgRNA载体0.8μg(每个转染体系中一种CRISPR/Cas9-sgRNA载体)，precutpUCA(LUC)-ROSA26-1载体0.2μg。转染6h后更换新鲜培养基，继续培养24h，吸取50μl培养上清至白色不透明96孔板中，按照Promega公司Renilla Luciferase Assay操作说明书配置LUC反应液(10μl的100X Renilla Luciferase Assay Substrate与1ml的RenillaLuciferase Assay Buffer混合得到)，向每孔加入LUC反应液50μl，立即使用96孔发光仪检测luciferase信号，计算平均值。并与Control组比较，分析各种sgRNA的活性。其中，Control组为试剂盒提供的阳性对照。
结果显示，各组CRISPR/Cas9-sgRNA的均具有较好的切割活性，其中CRISPR/Cas9-sgRNA1、2、3、4、6和8的剪切效率更为显著(图1)。
五、靶位点扩增测序法测定sgRNA的活性
传代猪胎儿成纤维细胞(所用细胞培养基为含10％FBS的DEME培养基)至24孔板，次日用脂质体转染法转染上述8个CRISPR/Cas9-sgRNA载体(其中，每个转染体系含有Lipofectamine 2000转染试剂2μl，CRISPR-Cas9-sgRNA载体1μg，每种体系含有一种CRISPR-Cas9-sgRNA载体)，转染72小时后提取细胞基因组DNA，并利用引物对1(CCTGAAGGACGAGACTAGCTCTAC和TCTGACCGTAAGGATGCAAGTGAG)对各sgRNA的靶序列区域进行PCR扩增与测序，分析各sgRNA靶序列突变情况。测序情况见表3。
表3测序分析各sgRNA引导Cas9切割目标DNA的切割效率
结果显示，各sgRNA引导Cas9切割目标DNA的体系中，含有sgRNA6、1和2的体系中DNA的编辑效率最高，均超过了50％，其余体系中，含有sgRNA7、5和4、8以及3的体系中DNA的编辑效率依次降低。
六、Surveyor酶切法测定不同sgRNA的Cas9剪切效率
所用试剂盒为SURVEYOR MUTATION DETECTION KIT,TRANSGENOMIC公司，CAT.706020，LOT.130421。
传代猪胎儿成纤维细胞(所用细胞培养基为含10％FBS的DEME培养基)至24孔板，次日用脂质体转染法转染上述CRISPR/Cas9-sgRNA载体(其中，每个转染体系含有Lipofectamine 2000转染试剂2μl，CRISPR/Cas9-sgRNA载体1μg，每种体系含有一种CRISPR-Cas9-sgRNA载体)，转染72小时后提取细胞基因组DNA，并利用引物对1(CCTGAAGGACGAGACTAGCTCTAC和TCTGACCGTAAGGATGCAAGTGAG)对各sgRNA的靶序列区域进行PCR扩增，对各PCR产物进行Surveyor酶切，3％凝胶电泳分析酶切结果。
结果显示，转染CRISPR/Cas9-sgRNA2的体系中酶切后的两条电泳条带最亮，表明，sgRNA2引导Cas9切割目标DNA时的效率最高，可以利用sgRNA2引导Cas9实现ROSA26基因的编辑。部分结果见图2。
实施例2、利用实施例1的靶序列2构建ROSA26定点整合转Sfat1基因猪
一、重组载体的构建
1、pCRISPR/Cas9-ROSA-sg2载体的构建
合成上下游寡核苷酸5’-CACCGCTCCTTCTCGATTATGGGCGGG-3’和5’-AAACCCCGCCCATAATCGAGAAGGAGC-3’，用ddH2O溶解至100μM，分别取上下游互补序列溶液15μl混合，置于95℃变性5min，而后以0.2℃/s的速度匀速降温至16℃，得到sgRNA2的靶DNA双链寡核苷酸。用NEB公司的BsmB I酶切CRISPR/Cas9载体(百奥赛图基因生物技术有限公司的UCA CRISPR/Cas9快速构建及活性检测试剂盒中载体)，将得到的载体骨架与sgRNA2的靶DNA双链寡核苷酸相连，将得到的序列正确的重组载体命名为pCRISPR/Cas9-ROSA-sg2(即实施例1中的CRISPR/Cas9-sgRNA2)，pCRISPR/Cas9-ROSA-sg2能表达靶向实施例1中靶序列2的sgRNA2与Cas9。
2、供体模板载体(pLoxP-Sfat1)的构建
将pEasy-Sfat1载体(Zhou Y,Lin Y,Wu X,Feng C,Long C,Xiong F,Wang N,PanD,Chen H.The high-level accumulation of n-3polyunsaturated fatty acids intransgenic pigs harboring the n-3fatty acid desaturase gene fromCaenorhabditis briggsae.Transgenic Res.2014，23(1):89-97.)的EcoR I和Sac I识别序列间的DNA片段替换为pCAGGS载体的EcoR I和Sac I识别序列间的DNA片段，获得pCAGGS-Sfat1载体。将pCAGGS-Sfat1载体用Hind III和Sal I酶切并补平粘末端，获得包含CAGpromoter和兔β-globulin polyA的Sfat1表达框架。将pLoxP-neo载体(Zhou Y,Lin Y,WuX,Feng C,Long C,Xiong F,Wang N,Pan D,Chen H.The high-level accumulation of n-3polyunsaturated fatty acids in transgenic pigs harboring the n-3fatty aciddesaturase gene from Caenorhabditis briggsae.Transgenic Res.2014，23(1):89-97.)用Xho I酶切并补平粘末端，将Sfat1表达框架插入其内，将获得的序列正确的重组载体命名为pLoxP-Sfat1-neo载体。用Q5高保真酶从猪基因组中扩增sgRNA靶向的ROSA26基因两侧各1700bp左右的序列，作为同源臂，并在左同源臂的两端分别加入Not I和Hpa I位点，右同源臂的两端分别加入Sal I和Kpn I位点，依次将左、右同源臂插入pLoxp-Sfat1-neo载体的相应位点，将得到的序列正确的重组载体命名为pLoxP-Sfat1(结构示意图见图3)，作为供体模板载体。pLoxP-Sfat1中，左右同源臂以及二者间的Sfat1表达框架的序列如序列表中SEQ ID NO.9所示。
其中，SEQ ID NO.9的第1-8位为Not I的识别序列，第9-998位为左同源臂序列，第998-1003位为Hpa I的识别序列，第1400-2641位为CAG启动子的序列，第2744-3943位为Sfat1的基因序列，第4030-4442位为兔β-globulin polyA的序列，第4504-4538位为loxP的序列，第4564-5070位为PGK启动子序列，第5081-5884位为neo基因序列，第6375-6408位为loxP的序列，第6414-6419位为Sal I识别序列，第6420-7970位为右同源臂序列，第7987-7970为Not I的识别序列。
SEQ ID NO.9的第2744-3943位所示的Sfat1的基因序列编码SEQ ID NO.10所示的Sfat1蛋白质。
分别利用不同的限制性内切酶对pLoxP-Sfat1进行酶切鉴定，结果如图4所示，结果显示得到的正确的重组载体pLoxP-Sfat1。
二、ROSA26位点定点整合Sfat1基因单细胞克隆的筛选与鉴定
猪胎儿成纤维细胞生长到汇合度≥80％后，用胰蛋白酶将细胞消化下来并进行精确计数，1 000r/min离心5min，离心后去上清液，用D-PBS重悬细胞洗涤1次再离心，尽量将上清去除干净，然后用37℃温育的100μL预混有8μg载体的Lonza转染液(该转染液中pCRISPR/Cas9-ROSA-sg2与pLoxP-Sfat1的摩尔比为1：3)重悬1×106个细胞，充分混匀后，转移至电击杯中，采用NucleofectorTM核转仪电击，电击程序为T016，电击结束以后，将细胞接种至60mm细胞培养皿中，用含有20％胎牛血清的DMEM培养48小时，转至含有400μg/mlG418和20％胎牛血清的DMEM中培养48小时，再换至含400μg/ml G418和2μM Ganciclovir和20％胎牛血清的DMEM中培养至细胞克隆点出现。
克隆点用记号笔标记，经D-PBS洗涤1次后，将灭菌的不锈钢克隆环放置于克隆点上，加入1滴胰蛋白酶消化3分钟，消化完成后加入全培养基终止消化，将克隆环内的液体(含有消化后的细胞)吸出，转入48孔细胞培养板进行培养，细胞长满后取一半用于鉴定，剩余细胞冻存。
提取细胞基因组DNA，进行PCR检测。利用引物对2(5’GCATATCGTTTGTTACGCTGGAAG3’和5’GGAAAGTCCCATAAGGTCATGTAC 3’)鉴定5’端是否发生同源重组，正确同源重组能扩增到大小为1381bp的DNA片段；利用引物对3(5’CAGCCTCTGTTCCACATACACTTC 3’和5’AGCCATACTTCCAAGGCTCAACAG 3’)鉴定3’端是否发生同源重组，正确同源重组能扩增到大小为1719bp的DNA片段；利用引物对4(5’GTGCTGGTTATTGTGCTGTCTCATC 3’和5’GCGTGGTGAAGTTTGTGACTCTTTTG3’)鉴定Sfat1基因是否整合至细胞基因组中，正确同源重组能扩增到大小为530bp的DNA片段。
选取发生正确同源重组的阳性克隆细胞，解冻到48孔板中，正常培养，待细胞长至汇合率达80％时，用Cre腺病毒处理48小时。胰酶消化离心，收集细胞，一部分细胞提取细胞基因组DNA，剩余的细胞加入显微操作液，重悬后室温放置1h然后转到4℃备用(4℃存放不宜超过2h)。然后利用引物对5(5’AGCTGTCCCTCTTCTCTTATGGAG 3’和5’AGCCATACTTCCAAGGCTCAACAG 3’)及引物对6(5’CAGCCTCTGTTCCACATACACTTC3’和5’AGCCATACTTCCAAGGCTCAACAG 3’)PCR鉴定Neo基因是否被删除，Neo基因未被删除引物对5能扩增得到大小为1723bp的DNA片段，引物对6能扩增得到大小为3653bp的DNA片段，Neo基因删除后，引物对5不能扩增到DNA片段，引物对6能扩增得到大小为1693bp的DNA片段。Neo基因被删除的细胞作为核供体细胞用于核移植。图5。
三、ROSA26基因位点转Sfat-1基因猪的构建与鉴定
用带有12#针头的注射器，吸取猪(北京安定种猪场)卵巢上发育良好、大小适中(3～6mm)的卵泡中的液体，注入50mL离心管中。37℃自然沉淀后弃上清，冲卵液洗两遍。体视显微镜下挑取胞质致密、卵丘包裹3层以上的卵母细胞。用DMEM清洗3遍后，转入已在38℃培养箱中孵育4h的IVM1培养液(IVM1培养液为向NSCU23培养液中按10％的pFF，10IU/mLPMSG，10ng/mL EGF和10IU/mL的hCG浓度添加相应物质得到的培养液，充分混匀，其中NCSU-23培养液的配方为:300mL Milli-Q超纯水中依次加入3.178g NaCl，1.053g NaHCO3，0.178g KC1，0.081g KH2PO4，0.147g MgSO4·7H2O，0.125g CaCl2·2H2O，0.500g D-Glucose，0.073g Glutamine，0.438g Taurine，0.273g Hypotaurine，0.025g链霉素，0.033g青霉素粉末,将pH值调整到7.2～7.4之间，最后用超纯水定容至500mL，定容后的渗透压为295～310mOsm。用Millipore公司SVGP01050型0.22μm滤器进行除菌过滤后，保存于4℃)中，进行20h的体外成熟后，将卵母细胞转移至无PMSG及hCG的成熟液IVM2(成熟液IVM2为向NSCU23培养液中按10％的pFF和10ng/mL EGF添加相应物质得到的培养液)中继续培养，进行体外成熟。经过累计40小时左右的体外成熟，将卵母细胞转至含有浓度为1mg/ml透明质酸酶的DMEM的培养皿中，用200μL的移液器在显微镜下用吹打卵母细胞，待卵丘细胞完全脱落后，在体式显微镜下用实心玻璃针，将卵膜形态完整、卵周隙大小合适且有第一极体排出的成熟卵母细胞，放入显微操作液中备用。
将成熟的卵母细胞在显微镜下用持卵针吸牢，然后用注射针拨动卵母细胞，将卵母细胞上成熟的第一极体调整至时钟1点钟的位置；用注射针吸取第一极体以第一极体内侧的相邻的10％～20％的卵母细胞的胞质，去除卵母细胞细胞核；挑选直径20μm左右的，圆形、光滑的步骤二得到的核供体细胞，放入卵周隙，用注射针点压透明带，使供体细胞与受体卵的胞膜接触紧密，即得到重构卵；将重构卵加入到含有BSA的显微操作液(含有BSA的显微操作液的制备方法如下：0.770g NaCl，0.0356g NaHCO3，0.0296g MgSO4-7H2O，0.0162gKCl，0.0296g KH2PO4，0.0146g L-谷氨酰胺，0.1g葡萄糖溶解于60毫升超纯水中，充分混匀溶解后，添加0.238g HEPES，0.150g牛磺酸，0.0065g青霉素，0.4g牛血清白蛋白，0.005g硫酸链霉素，充分溶解后调整pH在7.2-7.4范围，定容于100毫升容量瓶，0.22μm过滤器除菌分装，置于4℃保存)中恢复5分钟；然后用融合/激活液(融合/激活液的溶剂为水，溶质及其浓度分别为0.1mmol/L MgCl2，0.28mol/L甘露醇，0.5mmol/L HEPES，0.1mmol/L CaCl2以及0.01％(质量百分比浓度)PVA)对重构卵清洗三遍，然后将重构卵分批转移到融合/激活液中，洗涤3次，每次少量的放入已铺满融合液的1mm融合槽内，用实心玻璃针将重构卵中的细胞与融合槽垂直，再用BLS细胞融合仪施加一个100μs，140V的直流电脉冲诱导融合，之后采用7.5μg/ml的CHX进行4h左右化学辅助激活，得到将猪卵细胞的核替换为步骤二的核供体细胞的核的重构细胞。
在体外对获得的重构细胞进行培养，12h后挑选成功发育到1-2细胞期并且形态良好的重构胚进行胚胎移植。胚胎移植的受体为自然发情的初情期的大白母猪(北京安定种猪场)。胚胎移植的方法是通过手术法将受体母猪的卵巢拉出，查看受体母猪的发情状况，以卵泡发育较好，且即将排卵的母猪为受体进行移植。将输卵管轻轻拉出，将胚胎注入输卵管的深部，每头受体母猪移植200枚左右的重构胚。共计移植6头受体母猪，胚胎移植30天后，利用超声波妊娠检测仪检测受体猪怀孕情况，确认受体猪成功受孕。
受体母猪采用自然分娩的方式分娩转基因克隆猪。出生后一周之内采样出生小猪的耳组织进行转基因猪的PCR鉴定。利用引物对2鉴定5’端是否发生同源重组；利用引物对3鉴定3’端是否发生同源重组；利用引物对4鉴定Sfat1基因是否成功整合至基因组中。利用未转染的猪胎儿成纤维细胞基因组DNA作为阴性对照，利用阳性猪胎儿成纤维细胞基因组DNA作为阳性对照。
阴性猪和阳性猪的鉴定结果如图6所示。结果表明，利用本发明的方法成功得到了ROSA26基因敲除猪。
<110> 中国人民解放军军事科学院军事医学研究院
<120> 用于突变ROSA26基因的系统及其应用
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<170> PatentIn version 3.5
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gcggccgcga ggcaggcggg agtgcggccc gccctgcggc aaccggaggg ggagggagaa 60
gggagcggaa aagcctggaa tacggacgga gccattgctc ccgcagaggg aggagcgctt 120
cctgctcttc tcttgtcact gattggccgc ttctcctccc gccgtgtgtg aaaacacaaa 180
tggcgtgttt tggttggagt aaagctcctg tcagttacag cctcgggagt gcgcagcctc 240
ccaggaactc tcgcattgcc ccctgggtgg gtaggtaggt ggggtggaga gagctgcaca 300
ggagggcgct gtcggcctcc tgcgggggga ggggagggtc agtgaaagtg gctcccgcgc 360
gggcgtcctg ccaccctccc ctccggggga gtcggtttac ccgccgcctg ctcggctttg 420
gtatctgatt ggctgctgaa gtcctgggaa cggccccttg ttattggctt gggtcccaaa 480
tgagcgaaac cactacgcga gtcggcaggg aggcggtctt tggtacggcc ctccccgagg 540
ccagcgccgc agtgtctggc ccctcgcccc tgcgcaacgt ggcaggaagc gcgcgcagga 600
ggcgggggcg ggctgccggg ccgaggcttc tgggtggtgg tgactgcggc tccgccctgg 660
gcgtccgccg cctgaaggac gagactagct ctacctgctc tcggacccgt gggggtgggg 720
ggtggaggaa ggagtggggg gtcggtcctg ctggcttgtg ggtgggaggc gcatgttctc 780
caaaaacccg cgcgagctgc aatcctgagg gagctgcagt ggaggaggcg gagagaaggc 840
cgcacccttc tccgcagggg gaggggagtg ccgcaatacc tttatgggag ttctctgctg 900
cctccttttc ctaaggaccg ccctgggcct agaaaaatcc ctccctcccc cgcgatctcg 960
tcatcgcctc catgtcagtt tgctccttct cgattatgtt aacctactcg atcgacattg 1020
attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 1080
ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 1140
ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 1200
ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 1260
tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 1320
tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 1380
cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc 1440
ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc 1500
gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 1560
gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat 1620
ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc 1680
tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct 1740
ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg 1800
taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg aaagccttga 1860
ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt gcgtgtgtgt 1920
gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc gctgcgggcg 1980
cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg ggggcggtgc 2040
cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg tgtgcgtggg 2100
ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc acccccctcc 2160
ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc gtggcgcggg 2220
gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc 2280
tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg gcggctgtcg 2340
aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg cgcagggact 2400
tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca ccccctctag 2460
cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg agggccttcg 2520
tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc cgcgggggga 2580
cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg 2640
ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa 2700
cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttcatggtcg ctcattcctc 2760
agacgggtta tccgccacgg ctccggtcac cggcggagat gttctggttg atgctcgcgt 2820
ttctattgaa gagaagccac cacgctcctt ggattcaact caacagtcta ctgaggagga 2880
gcgcgttcaa ttgccaactg tggatgcatt ccgtcgtgcc attccacccc attgcttcga 2940
acgcgatctc accagatctc tcagatattt ggtgcaagac tttgcggccc ttgcttttct 3000
ttactttgct cttcctgtct tcgaatactt tggacttgtc ggttatctgg catggaacgt 3060
attgatggga gtcttcggat tcgctttgtt tgttgtcgga cacgattgtc ttcatggatc 3120
attctcagat aatcaagttt tgaacgatat tattggacac atcgcattct ctcctctttt 3180
ctctccctat ttcccatggc aaaagagtca caaacttcac cacgctttca ccaaccatat 3240
tgacaaagat cacggacacg tttggattca agacaaagac tatgaaaaga tgccaacatg 3300
gaagaagctg ttcaatccaa tgccgttctc gggatggctc aaatggttcc ccgtttacac 3360
actgttcgga ttctgcgatg gatctcattt ctggccatac tcttctcttt tcgtccgtga 3420
ttctgagcgt gttcaatgcg tgatttctgc cacttgctgt gtcgcttgtg cctatgttgc 3480
tctcgcgatt gctggatcct attcaaactg gttctggtac tactgggttc cactttcctt 3540
ctttggatgc atgctcgtta ttgtcaccta tcttcaacac gctgatgaag ttgctgaggt 3600
ttacgaagct gatgagtgga gtttcgtgcg tggacaaacc cagactatcg atcgtttcta 3660
tggatttgga ttggatgaga ccatgcatca catcactgac ggacacgttg cccatcattt 3720
cttcaataag attccacatt accatctgat cgaagctact gaaggtgtga agaaggtttt 3780
ggagccactg ttcgagactc agtacggata caagtaccaa gtaaattacg acttcttcgt 3840
ccgcttccta tggttcaaca tcaagctcga ctatcttgtc cataagacta aaggaatcct 3900
tcaattccga acaactcttg aggagaaggc aaaagccaag taagagctca attcactcct 3960
caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa 4020
taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag 4080
catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt 4140
ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag 4200
tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg 4260
ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga 4320
aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta 4380
acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta 4440
ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt 4500
cgagataact tcgtataatg tatgctatac gaagttatgt cgagggcccc tgcaggtcaa 4560
ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc 4620
ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc 4680
ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc 4740
ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa 4800
gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta 4860
ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg 4920
ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga 4980
aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc 5040
tcctcttcct catctccggg cctttcgacc tgcagccaat atgggatcgg ccattgaaca 5100
agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg 5160
ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg 5220
cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc 5280
agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt 5340
cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc 5400
atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca 5460
tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc 5520
acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg 5580
gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgatgatct 5640
cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc 5700
tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc 5760
tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta 5820
cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt 5880
ctgaggggat cgatccgctg taagtctgca gaaattgatg atctattaaa caataaagat 5940
gtccactaaa atggaagttt ttcctgtcat actttgttaa gaagggtgag aacagagtac 6000
ctacattttg aatggaagga ttggagctac gggggtgggg gtggggtggg attagataaa 6060
tgcctgctct ttactgaagg ctctttacta ttgctttatg ataatgtttc atagttggat 6120
atcataattt aaacaagcaa aaccaaatta agggccagct cattcctccc actcatgatc 6180
tatagatcta tagatctctc gtgggatcat tgtttttctc ttgattccca ctttgtggtt 6240
ctaagtactg tggtttccaa atgtgtcagt ttcatagcct gaagaacgag atcagcagcc 6300
tctgttccac atacacttca ttctcagtat tgttttgcca agttctaatt ccatcagaag 6360
ctgactctag caatataact tcgtataatg tatgctatac gaagttatct agagtcgacg 6420
ggcgggattc ttttgccctg gcttaacctg attcttgggc gttgtcctgc aggggattga 6480
gcaggtgtac gaggacgagc ccaatttctc tatattccca cagtcttgag tttgtgtcac 6540
aaaataatta tagtggggtg gagatgggaa atgagtccag gcaacaccta agcctgattt 6600
tatgcattga gactgcgtgt tattactaaa gatctttgtg tcgcaatttc ctgatgaagg 6660
gagataggtt aaaaagcacg gatctactga gttttacagt catcccattt gtagactttt 6720
gctacaccac caaagtatag catctgagat taaatattaa tctccaaacc ttaggccccc 6780
tcacttgcat ccttacggtc agataactct cactcatact ttaagcccat tttgtttgtt 6840
gtacttgctc atccagtccc agacatagca ttggctttct cctcacctgt tttaggtagc 6900
cagcaagtca tgaaatcaga taagttccac caccaattaa cactacccat cttgagcata 6960
ggcccaacag tgcatttatt cctcatttac tgatgttcgt gaatatttac cttgattttc 7020
atttttttct ttttcttaag ctgggatttt actcctgacc ctattcacag tcagatgatc 7080
ttgactacca ctgcgattgg acctgaggtt cagcaatact cccctttatg tcttttgaat 7140
acttttcaat aaatctgttt gtattttcat tagttagtaa ctgagctcag ttgccgtaat 7200
gctaatagct tccaaactag tgtctctgtc tccagtatct gataaatctt aggtgttgct 7260
gggacagttg tcctaaaatt aagataaagc atgaaaataa ctgacacaac tccattactg 7320
gctcctaact acttaaacaa tgcattctat cttcacaaat gtgaaaaagg agttccctca 7380
gtggactaac cttatctttt ctcaacacct ttttctttgc acaattttcc acacatgcct 7440
acaaaaagta cttttctgct caagtcacac tgagttgatt gctatttacc aaaatcaaag 7500
taacattatc agatctctgt agggtggttc cctctggaat gctaccctcc atagtcctta 7560
cccttcaagt aaagagcatg aagactgaaa tatctcctct gtgatctgtc atcctttaag 7620
ccagaatccc ccataaaaaa gttagtattg ctttctcctg atcccatagc aggttgaatc 7680
atagcactta tcaggttgtt gtcattgctt gcttaaattc tcctaactat ttggagcttc 7740
ttgagggcac aggttcttgt tgagtcttgt acctaagcac ctagtatagt ccttgatgtc 7800
tagccaaccc taaataaaat gcagtgagtg acatgtagat gtctttataa ggtttgatag 7860
gttggtctct caaatagttc ttttgtatgt ttggtagtgc tctagattag cactggccag 7920
tataactctg atgatggaaa tgttctatag ctatgctgtc taatatggta cc 7972
<210> 10
<211> 399
<212> PRT
<213> 人工序列
<220>
<223>
<400> 10
Met Val Ala His Ser Ser Asp Gly Leu Ser Ala Thr Ala Pro Val Thr
1 5 10 15
Gly Gly Asp Val Leu Val Asp Ala Arg Val Ser Ile Glu Glu Lys Pro
20 25 30
Pro Arg Ser Leu Asp Ser Thr Gln Gln Ser Thr Glu Glu Glu Arg Val
35 40 45
Gln Leu Pro Thr Val Asp Ala Phe Arg Arg Ala Ile Pro Pro His Cys
50 55 60
Phe Glu Arg Asp Leu Thr Arg Ser Leu Arg Tyr Leu Val Gln Asp Phe
65 70 75 80
Ala Ala Leu Ala Phe Leu Tyr Phe Ala Leu Pro Val Phe Glu Tyr Phe
85 90 95
Gly Leu Val Gly Tyr Leu Ala Trp Asn Val Leu Met Gly Val Phe Gly
100 105 110
Phe Ala Leu Phe Val Val Gly His Asp Cys Leu His Gly Ser Phe Ser
115 120 125
Asp Asn Gln Val Leu Asn Asp Ile Ile Gly His Ile Ala Phe Ser Pro
130 135 140
Leu Phe Ser Pro Tyr Phe Pro Trp Gln Lys Ser His Lys Leu His His
145 150 155 160
Ala Phe Thr Asn His Ile Asp Lys Asp His Gly His Val Trp Ile Gln
165 170 175
Asp Lys Asp Tyr Glu Lys Met Pro Thr Trp Lys Lys Leu Phe Asn Pro
180 185 190
Met Pro Phe Ser Gly Trp Leu Lys Trp Phe Pro Val Tyr Thr Leu Phe
195 200 205
Gly Phe Cys Asp Gly Ser His Phe Trp Pro Tyr Ser Ser Leu Phe Val
210 215 220
Arg Asp Ser Glu Arg Val Gln Cys Val Ile Ser Ala Thr Cys Cys Val
225 230 235 240
Ala Cys Ala Tyr Val Ala Leu Ala Ile Ala Gly Ser Tyr Ser Asn Trp
245 250 255
Phe Trp Tyr Tyr Trp Val Pro Leu Ser Phe Phe Gly Cys Met Leu Val
260 265 270
Ile Val Thr Tyr Leu Gln His Ala Asp Glu Val Ala Glu Val Tyr Glu
275 280 285
Ala Asp Glu Trp Ser Phe Val Arg Gly Gln Thr Gln Thr Ile Asp Arg
290 295 300
Phe Tyr Gly Phe Gly Leu Asp Glu Thr Met His His Ile Thr Asp Gly
305 310 315 320
His Val Ala His His Phe Phe Asn Lys Ile Pro His Tyr His Leu Ile
325 330 335
Glu Ala Thr Glu Gly Val Lys Lys Val Leu Glu Pro Leu Phe Glu Thr
340 345 350
Gln Tyr Gly Tyr Lys Tyr Gln Val Asn Tyr Asp Phe Phe Val Arg Phe
355 360 365
Leu Trp Phe Asn Ile Lys Leu Asp Tyr Leu Val His Lys Thr Lys Gly
370 375 380
Ile Leu Gln Phe Arg Thr Thr Leu Glu Glu Lys Ala Lys Ala Lys
385 390 395
Claims (10)
1.用于突变ROSA26基因的CRISPR/Cas9系统，包括靶向ROSA26基因的sgRNA，所述sgRNA的靶序列为如下A1)、A2)或A3)：
A1)序列表中序列2的核苷酸序列；
A2)与A1)限定的DNA序列具有75％或75％以上同一性的由A1)衍生的DNA序列；
A3)在严格条件下与A1)限定的DNA序列杂交的由A1)衍生的DNA序列。
2.根据权利要求1所述的系统，其特征在于：所述系统还包括Cas9。
3.根据权利要求1或2所述的系统，其特征在于：所述系统还包括供体DNA，所述供体DNA含有ROSA26基因中位于所述靶序列上下游的两个DNA片段。
4.根据权利要求3所述的系统，其特征在于：所述供体DNA还含有Sfat1编码基因或Sfat1编码基因的表达盒。
5.用于突变ROSA26基因的CRISPR/Cas9系统，包括与权利要求1中所述sgRNA相关的生物材料，所述生物材料为如下B1)-B8)中的任一种：
B1)编码权利要求1中所述sgRNA的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体；
B4)含有B2)所述表达盒的重组载体；
B5)含有B1)所述核酸分子的细胞系；
B6)含有B2)所述表达盒的细胞系；
B7)含有B3)所述重组载体的细胞系；
B8)含有B4)所述重组载体的细胞系。
6.根据权利要求5所述的系统，其特征在于：所述系统还包括与Cas9相关的生物材料，所述生物材料为如下C1)-C8)中的任一种：
C1)编码Cas9的核酸分子；
C2)含有C1)所述核酸分子的表达盒；
C3)含有C1)所述核酸分子的重组载体；
C4)含有C2)所述表达盒的重组载体；
C5)含有C1)所述核酸分子的细胞系；
C6)含有C2)所述表达盒的细胞系；
C7)含有C3)所述重组载体的细胞系；
C8)含有C4)所述重组载体的细胞系。
7.根据权利要求5或6所述系统，其特征在于：所述系统还包括权利要求3或4中所述供体DNA。
8.权利要求1-7中任一所述系统或权利要求1中所述sgRNA的靶序列的下述任一应用：
Y1)在突变ROSA26基因中的应用；
Y2)在制备ROSA26基因突变动物细胞中的应用；
Y3)在制备ROSA26基因突变动物中的应用；
Y4)在制备表达Sfat1动物细胞中的应用；
Y5)在制备表达Sfat1动物中的应用；
Y6)在制备富含不饱和脂肪酸动物中的应用；
Y7)在动物育种中的应用。
9.根据权利要求8所述的应用，其特征在于：所述动物为哺乳动物。
10.根据权利要求9所述的应用，其特征在于：所述哺乳动物为猪。
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