CN110172478A - CRISPR/Cas9系统介导的山羊KRTAP13-1基因敲除的方法 - Google Patents
CRISPR/Cas9系统介导的山羊KRTAP13-1基因敲除的方法 Download PDFInfo
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Abstract
本发明利用CRISPR/Cas9系统介导完成山羊KRTAP13‑1基因定点敲除，本研究依据山羊的KRTAP13‑1基因序列，构建基于CRISPER/Cas9系统的Cas9/gRNA打靶载体；然后将优化后的Cas9/gRNA共表达敲除载体转染至山羊胎儿成纤维细胞中，获得KRTAP13‑1基因定点敲除的细胞株。本发明所构建的基于CRISPER/Cas9系统的敲除载体为山羊KRTAP13‑1基因定点敲除提供了一种简单快捷安全的途径。该方法在细胞株筛选过程中没有涉及任何筛选标记基因，从而大大提高了转基因动物的安全性，对山羊的基因功能以及遗传育种的研究具有重要价值。
Description
技术领域
本发明涉及分子生物学及动物遗传育种领域，具体地说，涉及 CRISPR/Cas9技术介导的山羊KRTAP13-1基因定点敲除的方法。
背景技术
在目前的生命科学领域内，CRISPR/Cas系统作为第三代基因编辑工具展现出巨大的潜力和前所未有的影响力。在动物学、植物学和医学等领域都引起了巨大的变革，也得到了充分的肯定。在CRISPR/Cas 系统出现之前，对于基因的定点修饰作用主要通过锌指核酸酶(ZFN) 和转录激活因子效应物(TALE)实现。但这两项技术仅限于某些模式生物并且效率较低，还需要对每个靶位点进行复杂的蛋白质工程，这也大大增加了实验难度和成本。CRISPR/Cas9系统不同于第一代的ZFN 和第二代的TALE技术，它是一种使用互补RNA引导序列实现对目标位点精准识别的基因编辑过程，而且Cas9蛋白是一类不需要与其他蛋白质融合表达的核酸酶，只需要合成与靶序列互补的引导RNA分子就可以编辑几乎所有的基因位点。正是由于这种特性，大大简化了实验设计难度，使得基因编辑技术得到了前所未有的广泛应用。
内蒙古白绒山羊是我国特有的优秀山羊品种，有着饲养简单、育成时间短、绒肉兼用等特点，主要分布于内蒙古西部地区，是目前世界上产绒量最高、绒纤维品质最好的山羊品种。其产绒量约占中国的一半以上，占世界羊绒产量的1/3。而山羊绒作为绒山羊最重要的经济组成部分，是由山羊次级毛囊生长的珍贵绒毛纤维，又因为产量稀少导致价格十分昂贵。其中衡量羊绒品质和经济价值的核心指标就是羊绒细度。但目前在羊绒产业中对羊绒细度性状的维持和保护却远远不够。故培育超细绒型的绒山羊新品系显得十分重要。相对于传统育种，现代生物技术极大程度地缩短了育种所需时间，农业生物新品种培育提供了新的技术途径。
角蛋白是生物体内一种重要的结构蛋白，是毛发等皮肤衍生物的主要组成成分，对毛囊发育与毛发结构起到重要的作用。角蛋白家族又由角蛋白关联蛋白(KAP)和角蛋白中间丝蛋白(KIF)组成。其中 KIF基因的编码序列和结构基础在进化过程中相对保守，物种间差异并不显著。而KRTAP基因则在不同物种和相同物种不同种系间都存在显著差异，是决定毛发结构和性状的主效基因家族。目前，依据氨基酸一致性和相关含量将KRTAP家族划分为三大类：高硫KRTAP家族、超高硫KRTAP家族和高甘氨酸/酪氨酸KRTAP家族。其中高硫KRTAP 家族中的KRTAP13-1基因，在山羊、绵羊与人类的序列分析中存在高度同源性。并且在前期研究中发现，在人类毛囊中，KAP11-1和KAP13-1 是分布范围最广泛的两种KAP。同样对绵羊毛囊蛋白质组学研究表明， KAP11-1和KAP13-1是在毛囊形态发生过程中最早表达的两种KAP。这两种KAP可能参与了毛囊的构成并进而影响了毛发性状。虽然， KRTAP13-1基因已经被证实与绒毛纤维直径性状存在密切联系。在中国的一些绵羊和山羊品系中：新吉细毛羊、西藏绒山羊、博格达绒山羊、内蒙古绒山羊和辽宁绒山羊中均发现KRTAP13-1基因的多态性变化对于羊绒纤维性状存在着较大的影响。但是现在并未有研究揭示 KRTAP13-1基因对于毛囊结构和发育的具体作用机制，故而探究 KRTAP13-1基因对毛囊的研究工作有着重要意义。
利用CRISPER-Cas9系统敲除山羊KRTAP13-1基因的研究未见报道
发明内容
发明的目的是提供CRISPR/Cas9系统介导山羊KRTAP13-1基因定点敲除的方法。
为了实现本发明目的，本发明提供的CRISPR/Cas9系统介导山羊 KRTAP13-1基因定点敲除的方法，其是依据山羊KRTAP13-1基因序列 (Gene ID：102181431)，在KRTAP13-1基因的外显子上设计靶序列，构建基于CRISPR/Cas9系统的Cas9/gRNA共表达载体。然后将优化后的 Cas9/gRNA共表达转入山羊胎儿成纤维细胞中，获得山羊KRTAP13-1基因定点敲除的细胞株。
本发明中述及的山羊包括但不限于阿尔巴斯绒山羊。
前述的方法，gRNA作用靶位点位于山羊KRTAP13-1基因外显子上。 sgRNA作用位点的DNA序列为5′-CAGGACCACCTGCGCTACTC-3′。
前述的方法，所述的Cas9/gRNA共表达载体的核苷酸序列如SEQ ID NO:1所示(Cas9/gRNA表达载体图谱见图1)；
本发明还提供根据上述方法获得山羊KRTAP13-1基因定点敲除的细胞株。
本发明进一步提供上述方法在制备KRTAP13-1基因定点敲除的体细胞克隆山羊的应用。该应用是指将所述山羊KRTAP13-1基因定点敲除的细胞为核移植供体细胞，离体的山羊卵母细胞为核移植受体细胞，通过体细胞核移植技术获得山羊克隆胚胎，然后将克隆胚胎通过胚胎移植技术移入受体山羊子宫内进行妊娠，获得KRTAP13-1基因定点敲除的山羊。
本发明的目的还可以采用以下的技术措施来进一步实现。
1)Cas9/gRNA共表达载体的优化；2)根据山羊的KRTAP13-1基因序列，设计基于CRISPER/Cas9系统的sgRNA作用位点；3)将上述优化的Cas9/gRNA共表达载体转染到山羊胎儿成纤维细胞中，通过流式细胞仪分选法和口吸管分选法筛选单克隆细胞系；4)通过PCR技术鉴定筛选单克隆细胞株，获得KRTAP13-1基因定点敲除的单克隆细胞株。
其中，步骤3)中利用口吸管分选法筛选单细胞的操作为：预先将细玻璃管在酒精灯上拉制成足够细的口吸用玻璃针管，用75％的酒精擦拭所用的体视镜和显微镜，挑选电转染48h后状态良好、细胞活力好的细胞系，去除细胞培养废液，然后用PBS缓慢冲洗一遍并弃尽，用2mL胰蛋白酶直接作用于皿底，用移液器将皿中液体轻轻吹匀并在 37℃的5％CO2细胞培养箱中放置90s，立即用5mL含有10％FBS的细胞培养液停止消化过程。利用培养液稀释到合适的浓度，用新拉制的口吸用玻璃针管将细胞逐一打入到96孔板中，并放置于37℃的5CO2细胞培养箱中培养10d。
其中，步骤3)中利用流式细胞仪分选法筛选单细胞的操作为：挑选电转染48h后状态良好、细胞活力好的细胞系，去除细胞培养废液，然后用PBS缓慢冲洗一遍并弃尽，用2mL胰蛋白酶直接作用于皿底，用移液器将皿中液体轻轻吹匀并在37℃的5％CO2细胞培养箱中放置90s。立即用5mL含有10％FBS的细胞培养液停止消化过程，制成细胞悬液。离心后，用2mLPBS重悬细胞沉淀并收集于15mL离心管中，利用流式细胞仪的分选功能将单一细胞打入到每孔含有200μL培养液的96孔板中，在37℃的5CO2细胞培养箱中培养10d。
步骤4)中鉴定KRTAP13-1基因敲除单克隆细胞系主要采用设计扩增包含靶位区域的PCR引物的方法，通过PCR技术扩增靶序列区域的全部序列再对该扩增片段进行测序分析，目的是确定KRTAP13-1基因的突变情况。
本发现共获得100株单克隆细胞系，其中17株实现KRTAP13-1单等位基因敲除，敲除效率为17％；3株实现KRTAP13-1双等位基因敲除，敲除效率为3％。具体突变类型见图6。
本发明具有以下优点。
(一)CRISPR/Cas9基因编辑技术与常规的同源重组技术、ZFNs 技术、TALEN技术相比，基因编辑效率显著提高。
(二)在KRTAP13-1基因上设计sgRNA，敲除KRTAP13-1基因后能有助于探究KRTAP13-1基因的作用机理和对毛囊直径的影响。
(三)通过CRISPR/Cas9系统的介导，实现了不添加任何筛选标记即可筛选出KRTAP13-1基因定点敲除的细胞株，这是传统同源重组技术、ZFNs技术和TALEN技术无法实现的，很大程度提高了转基因动物的安全性。
(四)通过体细胞核移植技术制备KRTAP13-1基因定点敲除山羊，为构建成熟基因修饰动物的研究和生产奠定基础。
附图说明
图1为本发明实施例1中hCas9质粒图谱。
图2为本发明实施例2中CRISPR/Cas9系统介导山羊KRTAP13-1基因定点敲除模式图。
图3为本发明实施例4中利用Surveyor突变检测试剂盒检测靶位点的电泳结果；其中，1:DL500bp Ladder marker；2至4:gRNA突变活性情况；6：阴性对照；7:阳性对照。
图4为本发明实施例5中KRTAP13-1基因定点敲除总细胞靶区序列测序双峰图。
图5为本发明实施例5中KRTAP13-1基因定点敲除单克隆细胞图。
图6为本发明实施例6中突变细胞株KRTAP13-1基因定点敲除类型图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular cloning:a laboratory manual,2001)，或按照制造厂商说明书建议的条件。
以下实施例中测序工作由华大基因完成。
实施例1 Cas9-gRNA表达载体的优化
对购自北京唯尚立德生物科技有限公司的Cas9/gRNA共表达载体进行优化，优化后的Cas9/gRNA共表达载体的核苷酸序列如SEQ ID NO:1所示，质粒图谱见图1。
实施例2 Cas9/gRNA共表达载体的构建
根据山羊的KRTAP13-1基因序列(Gene ID：102181431)，基因序列如SEQ ID NO:2所示。在KRTAP13-1基因外显子序列上设计gRNA序列，并构建基于CRISPER/Cas9系统的Cas9/gRNA共表达载体。利用生物学软件根据sgRNA作用位点设计sgRNA序列，序列如SEQ IDNO:3所示。合成Target-Sense和Target-Anti，序列如SEQ ID NO:4、5所示，使其退火形成部分互补的oligo二聚体，后将oligo二聚体插入到Cas9/gRNA表达载体中，得到完整的Cas9/gRNA共表达载体，转化大肠杆菌Trans1-T1，涂板，9h后，挑取单菌落，摇菌，对菌液进行PCR鉴定，确定阳性后，保菌，送华大基因公司测序，将测序正确的单菌落接种于含Amp的LB 培养基中，37℃、220rpm过夜揺菌，提取质粒备用。
实施例3电穿孔法转染山羊胎儿成纤维细胞
在细胞汇合度达到100mm平面皿底的80％时，细胞数量和活性状态最为理想，进行细胞电转染。先沿平面皿壁缓慢去除细胞培养废液，用PBS沿皿壁缓慢加入后弃尽，用2mL胰蛋白酶将细胞消化到形态呈圆形但尚未从皿底成片飘起时，用5mL含有10％FBS的培养液停止消化过程。有移液器反复冲洗皿底后收集细胞悬液并1500rpm离心5min后，去除上清液。用opti-MEM电转缓冲液反复冲洗细胞沉淀三遍后，收集细胞沉淀用80μL opti-MEM重悬，加入Cas9/gRNA共表达载体质粒2μg，用无酶水将体积补齐至100μL，缓慢混匀后用移液器移入经灭菌的电转杯中，将电转仪条件设置为225V，2.5msec，进行电转染。电转染后将细胞置于37℃的5％CO2细胞培养箱中，在24h后进行换液处理，再24h后利用流式细胞仪分选法或者口吸管分选法将单细胞打入到96 孔板中继续培养。
实施例4 CRISPER/Cas9系统中sgRNA的活性检测
将Cas9/gRNA共表达载体通过电穿孔法转染山羊胎儿成纤维细胞，提取细胞的基因组DNA，以提取的基因组为模板跨靶序列设计的上、下游引物进行PCR扩增，扩增引物为K629F、K 629R，序列如SEQ ID NO:6、7所示。扩增体系(50μL)为：5μL 10X聚合酶缓冲液、4μLdNTPs、 2μL K629F、2μL K629R、1.5μL基因组、19.5μL 无酶水。反应条件为：94℃预变性5min；95℃变性30s、58℃退火30s， 72℃延伸50s，35个循环；72℃额外延伸10min，4℃保存。扩增得到大小为519bp的DNA片段，配制1％的琼脂糖凝胶，进行电泳检测，胶回收目的条带，胶回收产物逐步退火使其进行DNA杂交，杂交体系为：胶回收产物30μL，10×La PCR Buffer3μL。杂交程序见表1。
表1 DNA杂交程序
| 反应温度 | 反应时间 |
| 95℃ | 3min |
| 85℃ | 1min |
| 75℃ | 1min |
| 65℃ | 1min |
| 55℃ | 1min |
| 45℃ | 1min |
| 35℃ | 1min |
| 25℃ | 1min |
| 16℃ | Hold |
得到了杂交DNA后，通过用Surveyor突变检测试剂盒来检测突变活性。根据Surveyor突变检测试剂盒的使用说明书来完成实验，具体的反应体系见表2
表2 Surveyor突变检测体系
| 组分 | 体积 |
| 杂交DNA | 10μL |
| 0.15M Mgcl<sub>2</sub> | 1/10PCR产物体积 |
| Surveyor Nuclease S | 1μL |
| Surveyor Enclease S | 1μL |
| 组分 | 体积 |
将反应体系配置完毕后，置于42℃反应1小时，最后将入1μL的 Stop Solution后，将上述反应液通过2％琼脂糖凝胶电泳检测DNA片段的大小。检测结果见图3，sgRNA三个重复样品具有活性，均可以介导 Cas9对靶位序列进行切割。
实施例6单克隆细胞株的筛选
本实施例旨在筛选KRTAP13-1基因定点敲除单克隆细胞株。
提取CRISPR/Cas9表达载体。将从山羊胎儿分离得到的原代GFFs 解冻在100mm培养皿中培养，隔天换液。在细胞汇合度达到100mm 平面皿底的80％时，细胞数量和活性状态最为理想，进行细胞电转染。先沿平面皿壁缓慢去除细胞培养废液，用PBS沿皿壁缓慢加入后弃尽，用2mL胰蛋白酶将细胞消化到形态呈圆形但尚未从皿底成片飘起时，用5mL含有10％FBS的培养液停止消化过程。有移液器反复冲洗皿底后收集细胞悬液并1500rpm离心5min后，去除上清液。用opti-MEM电转缓冲液反复冲洗细胞沉淀三遍后，收集细胞沉淀用80μL opti-MEM重悬，加入Cas9/gRNA共表达载体质粒2μg，用无酶水将体积补齐至100μL，缓慢混匀后用移液器移入经灭菌的电转杯中，将电转仪条件设置为 225V，2.5msec，进行电转染。电转染后将细胞置于37℃的5％CO2细胞培养箱中，在24h后用胰蛋白酶消化细胞，用含血清的培养液终止消化，离心，弃掉上清，用PBS清洗细胞三次，务必洗净培养液。最后用少量PBS重悬细胞，通过流式细胞仪将单个细胞接种到预先加入含15％FBS DMEM/F12培养液的96孔板中。培养至10d左右时，观察细胞(见图5)，将96孔板中状态良好且长满孔底的单克隆细胞系去尽废液，用100μL胰蛋白酶作用90s后立即用移液器加入200μL含有10％FBS 的细胞培养液停止消化过程并反复缓慢冲洗孔底制成细胞悬液，移入到24孔板中，将细胞培养液体积补齐至1mL。待长满24孔板时制成细胞悬液，将500μL细胞悬液移入6孔板继续培养，500μL细胞悬液进行收样鉴定过程。得到鉴定结果后，将对应单克隆细胞系进行冻存处理。
实施例7单克隆细胞系的鉴定
以单克隆细胞株的基因组DNA作为模板，以包含靶位点的引物进行PCR扩增。具体的PCR反应引物为K29F、K29R，序列如SEQ ID NO:6、 7所示，反应体系(50μL)为：5μL 10X聚合酶缓冲液、4μL dNTPs、2μL K629F、2μL K629R、1.5μL基因组、19.5μL无酶水。反应条件为：94℃预变性5min；95℃变性30s、58℃退火30s，72℃延伸50s，35个循环； 72℃额外延伸10min，4℃保存。将PCR产物送往华大基因公司测序，结果如图6。经测序分析共有3株单克隆细胞株实现了KRTAP13-1基因的定点敲除。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 内蒙古大学
<120> CRISPR/Cas9系统引导的山羊KRTAP13-1基因定点敲除的方法
<130> CRISPR/Cas9系统引导的山羊KRTAP13-1基因定点敲除的方法
<141> 2019-04-29
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8360
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
aagtttcaat ggcgttccca aagggagtta agtcacgtat agaagcgaat atatattaaa 60
tacatatata tatataatat atatcaacct acatgctttc aataagatca caggccgggc 120
agtaaccttt gggctacagt cgctagcaag aaaatgattc aaggcctata aaaaatgcct 180
ttgatgaaag acacaatact agacaaaaag tttattcttt acaaatatag taaaatatgg 240
agtttcttaa accccctaaa ccgcagccat aaaaaaccta gcatacgccc ttgccttttg 300
tatacttcat cgctctggtc atacaacacc acatatctgt ggacggacgg acactccctt 360
gagcggacaa gaaaactgtc taatttggta gcaatttcga cagataaaag tcttcatcgc 420
tcactagtaa tgttcgtcga caaatatgcc atcgggaaga gtcagaaaac ctagaacctc 480
gctcactttg gaagagaaaa tggaggtaat ccagtcccag gagcgaaaca agctatcggt 540
tcgggatctg gccaagaggt aagatcaatg cctgatacat acatatgaat gggtaaaaat 600
aacaagtttc cgaaggttta acattggaaa gacacaagca gcagacattt tgaagcacaa 660
acagtcgatt aaagagggtc tgttgagtgg agaacttaag ttgaatcaga tgcggaggaa 720
tcccctctct cagcggggcg cccaaatcga tgagatgtgc tttgattggt tctcccgtgt 780
ccgtaccgag aatataccca tatcggggga aatggtgcgg aagaaggcca agcagttagc 840
tgtggagctg ggccactcca atttctctgc ctcctccgga tggctggaga aatggcgaaa 900
gcgacacaac gtccgataca atgacaccgg tgacagcctg gatctgcagg agttcgaggc 960
gattctggtg aaaagcgaac ccatctcgaa taaggacgat tgtgatgagc cataccccgt 1020
gactctgata gagcctatct attccaccga ggaggccatg atgcagctgg cccggctgaa 1080
ggagttcgca aaggatgact atgcttccta ccagcagttg atcagtctgg aaaatcaatg 1140
gagctggaaa tggaacatct ttaagaagga gcttccctga cccaagcatg caatttaggt 1200
gcaatataaa tacatattta tctcgaatct agtagtaaaa gttatttaat gtacgtttct 1260
tttataagta acatcgttga agataactta tcccttctac gcgttgaatt taaacctaaa 1320
actattgggt tattctattc atgacgatgc tcgtaaatat acgaaataca tgaccgtttg 1380
ctaatacgac tcactatagg caccttcctc ttcttcttgg ggtcagccct gcttacctgt 1440
tcttcatgag gtaacccgag ctatagccgt gtttgtcgca gccgacccgg cagtaatgcc 1500
tgctcatgtt ccacggctcg ttttttaagt ttcaagaccc gttatggcta gcggtgtcgt 1560
atttcttctt ggagtaaccg cgggaggaca agctgaggcc cctctgccgg cttcggtgcg 1620
ccgagttttc ttgtcgtgcc gcgtctatat gggcgtcttt cttagcctag acgatggacg 1680
tcctctagaa atcattactc taccgattcc acctactgag aaagaaggta tccgacctcc 1740
tcaggaaaaa ccacctcctc ctatttttcg tgctcgcggt gggttagaaa ccgttatagc 1800
acctgctcca ccgcatggta cttttcatgg gttggtatat agtagactcc ttcttcgaac 1860
atctgtcatg actattccga ctgaacgcca actagataga gcgcgaccgc gtatactagt 1920
ttaaagcccc tgtgaaggag tagctccccc tggacttggg tctgttgtcg ctacagctgt 1980
ttgagaaata ggttgaccaa gtctgaatgt tagtcgaaaa gcttctcttg ggctagttgc 2040
gtaggcctca actgcggttt cgttaggact cgcgatccga caggtttagg gccgccgagc 2100
ttttggagta gcgtgtcgag ggacccctct tcttcttgcc ggacaaacca ttagaatagc 2160
gggacagtga gcccgactgg gggttgaaat ttagattgaa gctggaccgg cttctacggt 2220
tcgaagttga ctcgtttctg tggatgctac tactagagct gttagacgac cgggtctagc 2280
cgctggtcat gcgtctggaa aaaaaccgcc gtttcttgga cagtctgcgg taagacgact 2340
cactataaga cgctcacttg tgcctctagt ggtttcgagg cgactcgcga tcatactagt 2400
tcgcgatact actcgtggtg gttctgaact gaaacgactt ccgggaacag tctgtcgttg 2460
acggactctt catgttcctt taaaagaagc tagtcagatt tttaccgatg cggcctatgt 2520
aactgccgcc tcgttcggtc ctccttaaaa tgtttaaata attcgggtag aacctttttt 2580
acctgccgtg gctcctcgac gaccatttcg aattgtctct tctagacaac gcgtttgtcg 2640
cgtgaaagct gttaccttcg tagggggtgg tctaagtgga cccgcttgac gtgcgatagg 2700
agtccgccgt tctcctaaag atggggaaaa actttctatt gtcccttttc taactctttt 2760
aggagtgtaa agcctatggg atgatacatc cgggggagcg ggccccttta aggtctaagc 2820
gcacctactg agcgtttagt cttctctggt agtgagggac cttgaagctc cttcagcacc 2880
tattcccccg gagacgggtc aggaagtagc tttcctactg attgaaacta tttttagacg 2940
gattgctttt ccacgaagga tttgtgagag acgacatgct catgaagtgt caaatattgc 3000
tcgagtggtt ccagtttatg cagtgtcttc cctactcttt cggtcgtaag gacagacctc 3060
tcgtcttctt tcgatagcac ctggaggaga agttctgctt ggcctttcaa tggcactttg 3120
tcgagtttct tctgataaag tttttctaac ttacaaagct gagacaactt tagtcgcctc 3180
acctcctagc gaagttgcgt agggaccctt gcatagtgct agaggacttt tagtaatttc 3240
tgttcctgaa ggacctgtta ctcctcttgc tcctgtaaga actcctgtaa caggagtggg 3300
aatgcaacaa acttctatcc ctctactaac ttcttgcgaa cttttgaatg cgagtagaga 3360
agctgctgtt tcagtacttt gtcgagttct ccgcggctat atgtcctacc cccgccgaca 3420
gttcttttga ctagttaccc taggctctgt tcgtctcacc tttctgttag gacctaaaag 3480
aattcaggct acctaaacgg ttggccttga agtacgtcaa ctaggtacta ctgagagagt 3540
ggaaattcct cctgtaggtc tttcgtgttc aaagaccggt ccccctgtca gaagtgctcg 3600
tgtagcgatt agaacgtcca tcgggtcgat agtttttccc ttatgacgtc tggcaattcc 3660
agcacctact tgagcagttt cattaccctt ccgtattcgg gctcttatag caatagctct 3720
accgggctct cttggtttga tgggtcttcc ctgtcttctt gtcatccctt tcctacttct 3780
cctaacttct cccatatttt cttgacccca gggtttagga attccttgtg ggtcaacttt 3840
tgtgggtcga agtcttactc ttcgagatgg acatgatgga cgtcttgccg tccctgtaca 3900
tgcacctagt ccttgacctg tagttagccg agaggctgat gctgcaccta gtatagcacg 3960
gggtcagaaa agagtttcta ctaagataac tattatttca caactgttct aggctatttt 4020
tatctccctt ctcactattg caggggagtc ttcttcaaca gttcttttac tttttaataa 4080
ccgccgtcga cgacttgcgg tttgactagt gtgttgcctt caagctatta gactgattcc 4140
gacttgctcc accggacaga ctcaacctat ttcggccgaa gtagttttcc gtcgaacaac 4200
tctgtgcggt ctagtggttc gtgcaccggg tttaagagct aagtgcgtac ttgtggttca 4260
tgctactttt actgtttgac taagctctcc actttcaata atgagacttc agattcgacc 4320
agagtctaaa gtctttcctg aaagtcaaaa tattccactc tctctagttg ttaatggtgg 4380
tacgcgtact acggatggac ttacgtcacc atccgtgacg tgaatagttt tttatagggt 4440
tcgaacttag acttaaacaa atgcctctga tatttcacat gctacaatcc ttttactagc 4500
gtttcagact cgtcctttat ccgttccggt ggcgattcat gaagaaaatg tcgttataat 4560
acttaaaaaa gttctggctc taatgtgacc ggttacctct ctaagccttc gctggtgaat 4620
agctttgttt gcctctttgt cctctttagc acaccctgtt cccatcccta aagcgctgtc 4680
aggccttcca ggacaggtac ggcgtccact tgtagcaatt tttctggctt catgtctggc 4740
ctccgaagag gttcctttca taggagggct tttccttgtc gctgttcgac tagcgtgcgt 4800
tttttctaac cctggggttc tttatgccgc ctaagctaag aggatgtcag cgaatgtcac 4860
atgaccaaca ccggtttcac ctctttccct tcagattttt tgagttttcg cagttccttg 4920
acgacccgta gtgttagtac ctcgctagtt cgaagctttt tttggggtag ctgaaagagc 4980
tccgctttcc tatatttctc cagttttttc tggagtagta attcgaaggg ttcatgagag 5040
agaaactcga acttttgccg gcctttgctt acgagcgatc acgcccgctc gacgtctttc 5100
cattgctcga ccgtgacggg agatttatgc aattaaagaa catagaccgg tcggtgatac 5160
ttttcgagtt tcccagaggg cttctattac tcgtcttcgt cgacaagcac cttgttgtgt 5220
ttgtgatgga actactctag tagctcgttt attcgcttaa gaggttttct cactaggagc 5280
ggctgcgatt ggagctattc cacgaaagac gaatgttatt cgtgtcccta ttcgggtagt 5340
ccctcgtccg tcttttgtaa taggtgaaca aatgagactg gttgaacccg cgcggacgtc 5400
ggaagttcat gaagctgtgg tggtatctgt ctttcgccat gtggagatgt ttcctccagg 5460
acctgcggtg tgactaagta gtcagttaat gccccgagat actttgttct tagctggaga 5520
gagtcgagcc acctctgtaa gatacattga tgagtttgga caaaccacaa ctagaatgca 5580
gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 5640
aagctgcaat aaacaagttt gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac 5700
tggatccggt accaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt 5760
gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa 5820
gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt 5880
aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt 5940
cttggcttta tatatcttgt ggaaaggacg aaacaccgtc gtgggggaga agttccgagt 6000
tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg 6060
caccgagtcg gtgctttttt tctagaccca gctttcttgt acaaagttgg cattagctcc 6120
cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg 6180
cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg 6240
tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 6300
gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 6360
cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 6420
taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg 6480
gtacccgggg atcctctaga gattatcgtc gacctgcagg catgcaagct tggcgtaatc 6540
atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 6600
agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 6660
tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 6720
aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 6780
cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 6840
ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 6900
ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 6960
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 7020
actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 7080
cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 7140
tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 7200
gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 7260
caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 7320
agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 7380
tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 7440
tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 7500
gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 7560
gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 7620
aagatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 7680
cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 7740
gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 7800
cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 7860
tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 7920
ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 7980
ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 8040
atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 8100
cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 8160
atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 8220
gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 8280
cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 8340
tctcgcggta tcattgcagc 8360
<210> 2
<211> 897
<212> DNA
<213> KRTAP13-1基因组序列(2 Ambystoma laterale x Ambystomajeffersonianum)
<400> 2
ctcagaatct tctcctagtc actcacctga actcacatca cctgcgaaga tgtcctacaa 60
ctgctgctct ggaaacttct cctcccgctc cctccaggac cacctgcgct actcaggctc 120
ctcctgtggc tcctccttcc ccagcaacct ggtctacagc actgacctct gctctcccag 180
ctcctgccag ctgggctcct ctctctacag ccaggagacc tgctgtgagc ccatcaggac 240
ccagactgtg gtgtcccgtc cctgccagac gtcctgctac cgcccgagga cctccacatt 300
ctccagtccc tgccagacaa ctttccctgg gtctctgggc tataggtcca gcagctgcag 360
ctccctgagc tctggatcca gaagctgcta cccagtgggc tgtggaggcc gtggcttcag 420
acgcctgggt tacggaatct gtggcttccc tgtcctgagc agtggatctg gattctgccg 480
gccaaccttc tttgcctcca ggagttgcca ctcttcctgt tataggccaa cctgtggatc 540
tgtcttctat tgatcagctt gagaagagtt cagatgtttt ctgcaatgtg tccaatctct 600
taacagagct tctatcatct tcatgatctt caggaagatt aagggtgcat cacatttttt 660
ccagtcatta aatacttacc aaaaatggtc tgccatgcat acttaatgat taactttatt 720
gattgattta ctgaatgcat taatggagtt agtaaaatga attcaatata ttactagatc 780
aattaatgaa atttatgtta agaaacaaat gaattaatgt tagtgaacta atggaatcct 840
tgttcatgta ttcacaaaat atataaaagc ctaaataaat ataataaact ctttcaa 897
<210> 3
<211> 20
<212> DNA
<213> sgRNA序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
caggaccacc tgcgctactc 20
<210> 4
<211> 28
<212> DNA
<213> target-sense序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
aaacaccgca ggaccacctg cgctactc 28
<210> 5
<211> 29
<212> DNA
<213> target-anti序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
ctctaaaacg agtagcgcag gtggtcctg 29
<210> 6
<211> 20
<212> DNA
<213> 上游引物序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
agccacagat tccgtaaccc 20
<210> 7
<211> 20
<212> DNA
<213> 下游引物序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 7
agccacagat tccgtaaccc 20
Claims (8)
1.CRISPR/Cas9系统引导的山羊KRTAP13-1基因定点敲除的方法，其特征在于，其是根据山羊的KRTAP13-1基因序列，构建基于CRISPER/Cas9系统的Cas9/gRNA共表达敲除载体，然后将优化后的Cas9/gRNA共表达敲除载体转染至山羊胎儿成纤维细胞中，获得KRTAP13-1基因定点敲除的细胞株。
2.根据权利要求1所述的方法，其特征在于，靶位点在KRTAP13-1基因的唯一外显子上。
3.根据权利要求2所述的方法，其特征在于，sgRNA靶序列为5′-CAGGACCACCTGCGCTACTC-3′。
4.根据权利要求1-3任一项所述的方法，其特征在于，所述山羊包括阿尔巴斯绒山羊。
5.根据权利要求1-4任一项所述的方法，其特征在于，所述CRISPER/Cas9系统中Cas9/gRNA共表达载体的序列如SEQ ID NO:1所示。
6.根据权利要求1-5任一项所述方法获得的山羊KRTAP13-1基因定点敲除的细胞株。
7.根据权利要求1-6任一项所述方法在生产KRTAP13-1基因定点敲除的克隆山羊中的应用。
8.根据权利要求7所述的应用，其特征在于，以权利要求6所述山羊KRTAP13-1基因定点敲除的细胞为核移植供体细胞，离体的山羊卵母细胞为核移植受体细胞，通过核移植技术获得山羊克隆胚胎，然后将克隆胚胎通过胚胎移植技术移入山羊子宫内妊娠，获得KRTAP13-1基因定点敲除的山羊。
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| CN106978445A (zh) * | 2017-06-12 | 2017-07-25 | 内蒙古大学 | CRISPER‑Cas9系统介导的山羊EDAR基因敲除的方法 |
| WO2018129341A1 (en) * | 2017-01-06 | 2018-07-12 | Alpine Biotherapeutics Corporation | Nucleic acids and methods for genome editing |
| US20180305719A1 (en) * | 2017-04-19 | 2018-10-25 | The Board Of Trustees Of The University Of Illinois | Vectors For Integration Of DNA Into Genomes And Methods For Altering Gene Expression And Interrogating Gene Function |
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| WO2018129341A1 (en) * | 2017-01-06 | 2018-07-12 | Alpine Biotherapeutics Corporation | Nucleic acids and methods for genome editing |
| US20180305719A1 (en) * | 2017-04-19 | 2018-10-25 | The Board Of Trustees Of The University Of Illinois | Vectors For Integration Of DNA Into Genomes And Methods For Altering Gene Expression And Interrogating Gene Function |
| CN106978445A (zh) * | 2017-06-12 | 2017-07-25 | 内蒙古大学 | CRISPER‑Cas9系统介导的山羊EDAR基因敲除的方法 |
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