CN108753835A - 一种利用CRISPR/Cas9编辑猪BMP15基因的方法 - Google Patents
一种利用CRISPR/Cas9编辑猪BMP15基因的方法 Download PDFInfo
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Abstract
本发明公开了一种利用CRISPR/Cas9编辑猪BMP15基因的方法，包括在目的猪基因组的BMP15基因的外显子1上设计两条gRNA，分别构建至pX458和pX459载体，使BMP15基因发生DNA片段的精确删除而丧失功能。与单条gRNA介导的编辑相比，本发明的方法中外显子DNA片段的精确删除能够更加有效地使BMP15基因丧失功能。
Description
技术领域
本发明属于生物技术领域，具体来说，涉及一种利用CRISPR/Cas9编辑猪BMP15基因的方法。
背景技术
骨形态发生蛋白15(Bone Morphogenetic Protein 15,BMP15)基因在小鼠，大鼠和人等动物的卵母细胞中特异性表达，具有促进颗粒细胞分裂增殖的作用，对早期卵泡发育具有十分重要的作用，是影响哺乳动物排卵率和产仔数的一个关键基因。BMP15已被鉴定为控制绵羊排卵数和多胎性状的一个主效基因，但关于BMP15基因对猪和牛等其他家畜繁殖性状的影响的研究报道相对较少。
不同物种中BMP15蛋白的作用不同，BMP15双等位基因敲除的小鼠仍具有产仔能力，但是BMP15双等位基因突变的绵羊丧失生殖能力，单等位基因突变的绵羊产仔数却会增加。该发现为利用BMP15基因突变提高家畜繁殖性状的研究提供了新的思路，即通过基因编辑技术结合体细胞核移植的方法，制备出BMP15基因编辑家畜，以期获得高繁殖力的BMP15基因编辑家畜。
Cas9和gRNA是CRISPR/Cas9系统的基本成分，gRNA用于特异位点识别，Cas9用于切割靶位点DNA。与传统的基因组编辑技术相比，CRISPR/Cas9系统的构建更加简便，快速，廉价。研究发现，当gRNA的靶位点位于同一条染色体上时，利用Cas9和多条gRNA共转细胞，可以产生两条gRNA靶位点之间DNA片段的删除，DNA片段删除能更有效地敲除目的基因。
发明内容
本发明的目的是针对以上要解决的技术问题，提供一种能够更加有效地敲除目的基因的利用CRISPR/Cas9编辑猪BMP15基因的方法。
为了实现以上发明目的，本发明提供了以下技术方案：
一种利用CRISPR/Cas9编辑猪BMP15基因的方法，其包括：在目的猪基因组的BMP15基因的外显子1上设计两条gRNA，分别将其构建至pX458载体和pX459载体，使所述BMP15基因的靶点切割区域的部分DNA片段被精确删除而终止表达，其中所述靶点切割区域的核苷酸序列如SEQ ID NO.6所示，其为如SEQ ID NO.1所示的核苷酸序列自5，末端第166-493位。
根据本发明的方法，其中被精确删除的部分DNA片段的核苷酸序列如SEQ ID NO.7所示，其为如SEQ ID NO.1所示的核苷酸序列自5，末端第299-470位。
根据本发明的方法，其中两条gRNA包括gRNA-E1S1和gRNA-E1S2，其均为单链DNA分子，gRNA-E1S1的核苷酸序列如SEQ ID NO.2所示，且gRNA-E1S2的核苷酸序列如SEQ IDNO.3所示。
根据本发明的方法，其中CRISPR/Cas9基因编辑包括如下步骤：
(1)将gRNA-E1S1构建到能表达Cas9蛋白和EGFP荧光蛋白的pX458载体上，得到pX458-gRNA-E1S1；将gRNA-E1S2构建到能表达Cas9蛋白和DsRed荧光蛋白的pX459载体上，得到pX459-gRNA-E1S2；将构建好的pX458-gRNA-E1S1和pX459-gRNA-E1S2共转染目的猪离体胎儿肾细胞，得到BMP15基因编辑细胞群；
(2)通过用于扩增BMP15基因的外显子1的靶点切割区域的引物对对所述BMP15基因编辑细胞群进行PCR扩增，通过T-A克隆方法检测PCR扩增产物，计算克隆中含有编辑型BMP15基因的克隆比例，即为CRISPR/Cas9系统编辑效率。编辑效率越高，获得BMP15基因编辑动物的比例越高。
根据本发明的方法，其中用于扩增所述靶点切割区域的引物对包括核苷酸序列如SEQ ID NO.4所示的单链DNA分子以及核苷酸序列如SEQ ID NO.5所示的单链DNA分子。
根据本发明的方法，其中编辑型BMP15基因为野生型BMP15基因外显子1中172bp大片段缺失使所述BMP15基因丧失功能所得到的基因型。
根据本发明的方法，其中野生型BMP15基因的核苷酸序列的核苷酸序列如SEQ IDNO.1所示。
根据本发明的方法，其中猪优选但不限于大白猪。
此外，本发明还提供了一种利用CRISPR/Cas9编辑BMP15基因的基因编辑动物的制备方法。
本发明提供的方法，为将上述方法制备的含有编辑型BMP15基因的胎儿肾细胞通过体细胞核移植获得BMP15基因编辑动物。
本发明还提供了一种研究BMP15基因对家畜繁殖力的作用的方法。
本发明提供的方法，为将上述方法制备的含有编辑型BMP15基因的胎儿肾细胞通过体细胞核移植获得BMP15基因编辑动物，制备出BMP15单等位基因编辑和BMP15双等位基因编辑的基因编辑动物，从而研究BMP15基因对于动物生殖力及产仔数的影响。
上述方法中，所述动物为哺乳动物，具体为猪，所述猪的品种优选但不限于大白猪。
本发明的实验证明，本发明在目的基因猪的BMP15基因的外显子1上分别设计两条gRNA，分别构建至pX458和pX459载体，使BMP15基因实现长片段DNA的更有效和精确的删除而终止表达，得到BMP15基因编辑猪。传统的提高猪繁殖力的方法为选取具有高繁殖力性能的亲本进行杂交选育，耗时较长，并且往往效益不高。本方法制备的BMP15基因型编辑猪，利用高效的CRISPR/Cas9系统以及核移植技术，可以快速得制备出BMP15单等位基因编辑和BMP15双等位基因编辑的基因编辑猪，通过进一步选育，即可得到高繁殖力的BMP15基因编辑猪。
附图说明
图1为gRNA-E1S1和gRNA-E1S2在猪BMP15基因上的位置示意图。
图2为猪胎儿肾细胞中gRNA-E1S1和gRNA-E1S2的删除效率鉴定。NC为阴性对照组，1、2泳道为未经流式分选的删除效率，3泳道为流式分选红绿双荧光后的删除效率。
图3为部分BMP15编辑猪测序结果图。
具体实施方式
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明，而非用于限制本发明的范围。
下述实施例中所使用的试验方法如无特殊说明，均为常规方法。
所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
猪的野生型BMP15基因的核苷酸序列为序列表中的SEQ ID NO.1。
猪的编辑型BMP15基因为上述野生型BMP15基因外显子1中172bp大片段(SEQ IDNO.7)缺失使猪BMP15基因丧失功能所得到的基因型。
针对BMP15基因外显子1所设计两条gRNA，当将其分别构建到能表达Cas9蛋白的pX458和pX459载体上，就形成了两种能够特异性识别BMP15基因并对识别位点进行打靶的CRISPR/Cas9系统。
这两种CRISPR/Cas9系统共转染细胞后，分别打靶BMP15基因上相应gRNA所识别的位点(对应SEQ ID NO.6)，从而删除BMP15基因上两条gRNA所识别位点的中间序列(对应SEQID NO.7)，实现猪BMP15基因上的大片段DNA的精确删除。
实施例1：
利用CRISPR/Cas9编辑猪BMP15基因，制备相关基因编辑细胞
1、离体猪胎儿肾细胞的获得
猪胎儿肾细胞从大白猪胎儿肾脏中分离，在超净台内进行猪胎儿肾细胞的分离。用剪刀和镊子取下胎儿的肾脏组织，将取下的组织依次在75％酒精以及添加了抗生素的PBS里反复清洗，用小剪刀将组织块剪至1立方毫米大小，1600rpm离心5min去除PBS，再加入带抗生素的20％FBS的DMEM，轻轻吹打均匀，放入37℃细胞培养箱培养。放入细胞培养箱后，不要挪动培养皿，三天后，可观察到猪胎儿肾细胞已爬满至整个培养皿，再进行一般传代细胞的消化培养即可。
2、含有编辑型BMP15基因的细胞的获得
1)质粒转染进细胞获得BMP15基因编辑细胞
针对猪BMP15基因外显子1所设计两条gRNA，分别构建到能表达Cas9蛋白的pX458和pX459载体上，形成两种能够特异性识别BMP15基因并对识别位点进行打靶的CRISPR/Cas9系统(如图1所示)。
设计用于编辑猪BMP15基因的两条gRNA序列如下：
gRNA-E1S1：5’-TGTACCTTGCCCCTGATTA-3’(SEQ ID NO.2)；
gRNA-E1S2：5’-TGGTGAGGCCATTGGTTAA-3’(SEQ ID NO.3)。
采用电转的方法将5μg pX458-gRNA-E1S1和5μg pX459-gRNA-E1S2共转染1*106猪胎儿肾细胞细胞。电转严格按照试剂盒和电转仪说明书操作。
2)流式分选EGFP和DsRed双阳性细胞
pX458-gRNA-E1S1和pX459-gRNA-E1S2载体转染进细胞后，分别表达EGFP绿色荧光和DsRed红色荧光，通过流式分选出的红绿双荧光细胞，即为携带pX458-gRNA-E1S1和pX459-gRNA-E1S2两种载体的细胞，这种双阳性细胞使BMP15基因发生DNA片段的精确删除而丧失功能。
3)鉴定含有编辑型BMP15基因的细胞
设计用于扩增删除区域的引物对如下：
BMP15-F：5’-CTGCCTGCCTTTCACTGTTT-3’(SEQ ID NO.4)；
BMP15-R：5’-TCCCTAGGCAAAGCAATTAGAT-3’(SEQ ID NO.5)。
将上述1)得到的编辑细胞基因组DNA作为模板，用上述BMP15-F与BMP15-R组成的引物对进行PCR扩增。野生型条带大小为569bp，删除后条带大小为397bp(如图2所示)。
实施例2：利用体细胞核移植技术构建BMP15因编辑猪
1、体细胞核移植获得BMP15基因编辑猪
从健康大白母猪体内采取挑选发育阶段适宜的卵巢，用注射器抽取卵巢表面直径在3-5mm的卵泡中的内含物，将内含物在TL-PVA中稀释并重悬形成悬浊液。将悬浊液在37℃环境下静置至卵母细胞沉淀完全，将沉淀吸出置于在体视镜下用移液器或口吸管挑选卵周细胞完整的卵母细胞。将挑选的健康卵母细胞放入含有10％卵泡液、FSH、LH、EGF的TCM-199中培养22h。再用移液器或口吸管将卵母细胞移到含有10％卵泡液、EGF的TCM-199中继续培养22h。经过44h培养成熟后挑选已经排出第二极体的健康成熟卵母细胞作克隆胚胎用。
将上述制备的大白猪的含有编辑型BMP15基因的细胞，于5％CO2、37℃饱和湿度的细胞培养箱培养，待细胞长至对数生长期时，即可用于核移植操作。
待卵母细胞体外培养成熟后，用采用电融合法将含有编辑型BMP15基因的细胞群进行体细胞核移植，并在24h之内进行胚胎移植，制备不同品种的BMP15基因型的基因编辑猪，BMP15编辑猪体细胞核移植及生产统计如表1所示。
表1
2、BMP15基因编辑猪的鉴定
采取少量BMP15基因编辑猪的耳组织样提取基因组作为模板，用BMP15-F与BMP15-R组成的引物对进行PCR扩增，并克隆测序，鉴定克隆猪的基因型。
测序结果表明，为对目的猪基因组的BMP15基因外显子1的靶点区域(序列1自5，末端第166-493位的核苷酸)利用CRISPR/Cas9进行基因组编辑，删除区域为序列1自5，末端第299-470位的核苷酸，使BMP15基因实现长片段DNA的精确删除而终止表达，得到BMP15单等位基因编辑和BMP15双等位基因编辑的基因编辑猪。部分BMP15基因编辑猪的鉴定结果如图3所示，共包含未编辑型、单等位基因编辑型和BMP15双等位基因编辑型三种类型，每个编辑猪样品挑取4至5个克隆进行测序统计。
序列表
<110> 中山大学
<120> 一种利用CRISPR/Cas9编辑猪BMP15基因的方法
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6337
<212> DNA
<213> 大白猪(Sus scrofa)
<400> 1
ctcttagaga aagcaacata ggacctgcct gcctgccttt cactgtttcc tggccccagc 60
ctttgtggta gtggagcctg ggtgctgtta cctgtgtgat agaaggcatc agagtggcat 120
cattttggct tgtgctggaa catgttgctg aacaagtctt tcaagatggt cctcctcagc 180
atcattagaa cccttcttct ttggggactg gtgcttttta tggaacacag ggtccaaatg 240
acccaggtag ggcaaccctc tgtggccctc ctgcctgagg cctgtacctt gcccctgatt 300
agggagctgc tagaggaagc ccctggcaaa cagcagagga agccacaggt cctggggcat 360
cccttgcgat atatgctgga gttgtaccag cgttcagccg acgcacgtgg gcaccctagg 420
gagaaccgca ccattggggc caccatggtg aggctggtga ggccattggt taatggagca 480
aggcctctca gaggtgagtt atcacacgat atggtgctgg aggggagaaa aatgagaaag 540
cagagtgtag agaaaagggg atctgttagc tgactatcta attgctttgc ctagggagtg 600
ggcggtttcc tcgggcttgg tttttgaagg atggccaact tgggagaagc tggctccaag 660
cctgcttccc tttagggccc tgatttaacg agagattgcc ttggggccta cctgaggaat 720
ctctcccagg ccccggggca actgagtgat gtgttagctc ctatctttca tagaaagtct 780
taatgcctgt tacatcacca aaaactacaa ttgagaaata cccatgtaca ttcctgtgga 840
gagcggtttg tgttgtttta ttattggaag gcaaggggta caggagggct gagttgggga 900
gatttacgtg gcctcagaaa ggacagaaac gggctaacat ctgagagagt ctgtttgcct 960
ggctccgtga ggatctgcta ttgtatttaa tccttaatga cagctactct cgttagcttt 1020
catggaacac ttagcctaat ttttaaaaat aagaaaacta gagcccagag acgttaaata 1080
atttgcagaa gtcgcaggga ccgactgggt ggcagagata ggatttgggc tccagtctgt 1140
ctgattctaa agtctttcca taagtactta agtgtttaaa tgtggtacct cgatatattt 1200
atataccaag tgatttttct atgtggactg tgttactact ttggggtttg ggggaccaaa 1260
tgtattttgg aattggcttt gttcccatca tagttgcttg ggctttaaac agcagcctta 1320
gacactgttc cagagcagta gcggattcct gaaatgcagg gctctctagg gagatgcagg 1380
cagccatcaa cttcaaggaa aatcttggga acctgttgat accaatctta agcctttgct 1440
agtcttagat gacaacagat aggatttacc ctccctcctc caagtaaaac caccaccatt 1500
cccctcagct aacatttact gtgagcttac cacatgctag gcattgggca aagtacttta 1560
caaacctcct taatccttcc ccactatgag gtaggtatta caaacatacc cattttatag 1620
gtgaagaaac tgaggctcaa ggtaacacag ctagtaattg atagatctgg gtttctaacc 1680
caggctttca ggcttctatg cttttaatta acctgctgtg ctgactggct taacagattt 1740
taattgagca gaaagcactg tgtttgggtt gggtataaag aggcaaataa ctgctaccct 1800
gacctctagg cactcccact gtagtcgaat ggatagagaa gtaattgcat ggtggtgtta 1860
tagtgtggca aatgctttga aaagaataag caataggggt gtcaggactc caaagaagaa 1920
atgtgttcct gtgcagagag acctttggaa ggtcccctca aggatgtggc atttgagctg 1980
gactttgtgg aaagagtaaa gagactatgc atatgggcgg cacattctga ttaaggaaaa 2040
agaaaataac ttggtgtggg tggagattcg ttcaacaaat atttatttgg caacttctaa 2100
gtgctagaca ttgttctagg tggcagtatt gggatttgat ccagatgggc atggttttca 2160
tgggtaagac aaataagcac tgaccagatt gtggtcagtg ctatcaaaga atgacataaa 2220
ttattttgat agaggacacc aaagaaagcc tcccttggtg tgggattgcc ggggagggtc 2280
acttgaagag gtgacatttg agctaaggtc tgtaaactaa ggagagggaa gtctgactga 2340
ggcagaggga acaagtgtgt aaaggcctaa agggtgaaaa ctgagcttgt ctgacttgaa 2400
gaacagaagg aagatcagga tggttggaac ctcatgaggt tgtggacagt ggcggagtat 2460
gaggctgatg agactcaagg ccttgtaggt tattaccggg gatctgggtt aagctgcaca 2520
agtcaattcc acgtagatga tatgaggcta gaaaggaggc tgcctagaat tgggccacta 2580
tcctgatctg actgcagggc ataacgggca tctaaggact ctggaaagga tccagaaatg 2640
gatgacaaga gagccaggcc tgatcctttt catcaggaga tgacccagct gaatcaagct 2700
ggcttggaga gctatcccaa gttcaatcac acaggatata tgagtcagcc atttccctga 2760
aaagttccga gcagaccctt cctggctctg atccttagca accccaccct cgtgcaggaa 2820
atatgtgatg tcagggtctg aaaggaaagg gcaattcctc accagacagc ctgctagaga 2880
ataggattcc tttttagtca gtgtgagaca gatgtatggg tgggcccctt ggaaagatta 2940
ttttgagaaa gtaaatataa gcctggcttg ggccttgtct gaatgatacc caagtgcctc 3000
caaacttaaa aggtgtatta agtaagcagg agctgggatg cgaactttaa aaaaaaaatt 3060
ctgatgtatg atttcactta tatgcagaat attaaaaaat gaccaaaacc caaataaaaa 3120
caaactcaga tacagagatc agattagtgg ttacgagcaa ggaagggggt tgggggtggg 3180
caaagtagat gaagggagtc aactctggta cagagatgat gataacgagg cttgtagtgg 3240
caatcacttc gtagtatata cagatgttgg tgctatacac ctgaaactta gaagaaactt 3300
agagacataa tataaaaagt atcctgatga gagttcccgc tctggcacaa cgggatgggc 3360
agcatctttg cagcgcaagg acacaggttt gatccctggc ccggcacagt gggttaaagc 3420
atctggcgtt tgctgccagc tgaggcttgg atctgatccc tggcttggga actgcatatg 3480
tcgtggggtg gccaaaaaaa gaaaaaaaaa tcttgatgtg tcaaaacatt aaaaggcttc 3540
taagttgacc atctcaggcc tgctgaagaa tttccgaccc agggatttag tggaaaaaaa 3600
aaaaggttaa gtaaataata tgtaacaaag acaaaatgtg tacatgcaat tgtctgcagc 3660
tgagtggaag gtaaggtagt agtttcacaa tgaaccagga tggtatatac caattgtcat 3720
tggcatcagt catgagtccc ttttgtttct atgaaagcag tagtgttgaa tcactacata 3780
ttaagtaatt acaacttgct ctattttggg attaaaacaa aattatcatt tctcctttga 3840
tggtactttc tttgaattat agtttcaaaa tccattagcc taggatgact tgatttggtc 3900
ataaataaga aaccaattga tagaagaaaa ctgattgtat ttcactgaaa tgttaacgaa 3960
atatcaccca aattggaacc atttggttta agagaaaatc aaaatattaa aattttctaa 4020
ttttaaattt tttacaaact cttctcatca gtctttactc atttttgtta taagtactta 4080
tttaatctag ttattaaaca ttaaaaataa gtttaaaata atatggaaat taaccgggca 4140
tgtttagtga tatttgaaga tgacagaagg cttttccttg ctatctgtag tttcttgtaa 4200
tcgtttctct tgacctttgc caggagaggg cgcagaaggt cagagcacta caggcccagc 4260
attccaaccc tttggccact gggtgtcagc atcacagttt agattaaaat gcttaatcat 4320
tgagaggcaa gcagctctga gtcaccttct tggcaaacaa aatatttata tgaccagggc 4380
ctgtttgaga atggtattta aggaaatggc agtagaatga atgggctaaa tgaagaatca 4440
tttccagtaa ggaatggaaa gaaaagggag agactgaggg ttaagggaga aacagcctaa 4500
gagggcaata gctgctcaag aagaaattta aatagagggc aaaaatggca aaatgcctgc 4560
cccccctagg ggcagataga caaaattatt ttgtggcatc tcggccctgg atttgaagtg 4620
atggcaggcc ataacctcta atggttgaac tgtcccctct aaagctatat ctatatagtg 4680
atgcaggatt tgttgttaac tcatgaatcg ctcaatgact ttcttcattg gtccccttta 4740
ataacacaga caatataatg atacaggcaa tatatataca tacaatttgg tatttgaggt 4800
gtttttcttg gcctagtgct gtaagtgatt taaaaaatga gacgcaattc tctttttaag 4860
cgaaaagact ttggctcaaa tcttgattcg aacacacacc tggctgtgtg tcctctggtg 4920
tagcctctcc gagcttcaat ttccttgtct tcaaaatggg aataataact atcacataag 4980
gctattgtgg attaaagagc taatgcatgg aaaacatctg acatattggt aagtgtgcca 5040
tatatgggag ctcttatgac gttgaattaa tctgagggct gcttctagct gtcagtttgt 5100
accgagtggg tttggtgggg gtggggagga gagtcaggct agaaggtaag aaattaatgc 5160
tctgttcttt tttttttttt tttttggtct ttttgccatt ttcttggacc gcttctgtgg 5220
tatatggagg ttcccaggct agggggtcga attggagctg tagctgccag cctacaccgg 5280
agccacagca acgcgggatc cgagtagcgt ctgccaccta catcacagct cacagcaacg 5340
ccggatcctc aacccaccga gtaaggccag ggatcgaacc tacaacctca tggtacctag 5400
tcggattcgc tagccactga gccacgatgg gaactccaat gctctgttct tgtttcctct 5460
tattcatgca gggccctggc atatacagac cttggacttt cctctgagac caaaccgggt 5520
agcctaccaa ctagtcagag ccactgtggt ttatcgccat caacttcacc tagctccctt 5580
ccacctctcc tgccatgtgg agccctggat ccagaaaagc acaaccagtc actttccttc 5640
ctcaggaaga ggctccttaa agccttccct gctgccccaa gcttggacgg agatggatgt 5700
cacgcaacat gttggacaaa agctctggaa tcacaagggg cgcagggttc tacgactccg 5760
cttcatgtgt cagcagcaaa atggtagtga gattcttgag ttccgggggc gtggcatttc 5820
atccctggac actgccttct tgttactcta tttcaatgac actcggagtg ttcagaaggc 5880
caaacttctt cccagaggcc tggaagagtt tatggcaaga gacccttctc ttcttttgcg 5940
gaaggcccgg caagcaggca gcatcgcatc tgaggttctt ggcccctcca gggagcacga 6000
tgggcctgaa agtaaccagt gttccctcca tcctttccaa gtcagcttcc accaactggg 6060
ttgggatcat tggatcattg ctccccattt ctatacccca aactactgta agggggtctg 6120
ccctcgggta ctacactatg gtctcaattc ccccaatcat gccatcatcc agaaccttgt 6180
caatgagctg gtggaccaga gtgtccctca gccctcctgt gtcccttata agtatgtgcc 6240
tattagcatc ctcctgattg aggcaaatgg gagtatcttg tacaaggagt atgaggatat 6300
gattgcccag ccctgtacat gcagatgaca gcaacag 6337
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgtaccttgc ccctgatta 19
<210> 3
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tggtgaggcc attggttaa 19
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ctgcctgcct ttcactgttt 20
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tccctaggca aagcaattag at 22
<210> 6
<211> 328
<212> DNA
<213> 大白猪(Sus scrofa)
<400> 6
atggtcctcc tcagcatcat tagaaccctt cttctttggg gactggtgct ttttatggaa 60
cacagggtcc aaatgaccca ggtagggcaa ccctctgtgg ccctcctgcc tgaggcctgt 120
accttgcccc tgattaggga gctgctagag gaagcccctg gcaaacagca gaggaagcca 180
caggtcctgg ggcatccctt gcgatatatg ctggagttgt accagcgttc agccgacgca 240
cgtgggcacc ctagggagaa ccgcaccatt ggggccacca tggtgaggct ggtgaggcca 300
ttggttaatg gagcaaggcc tctcagag 328
<210> 7
<211> 172
<212> DNA
<213> 大白猪(Sus scrofa)
<400> 7
ttagggagct gctagaggaa gcccctggca aacagcagag gaagccacag gtcctggggc 60
atcccttgcg atatatgctg gagttgtacc agcgttcagc cgacgcacgt gggcacccta 120
gggagaaccg caccattggg gccaccatgg tgaggctggt gaggccattg gt 172
Claims (8)
1.一种利用CRISPR/Cas9编辑猪BMP15基因的方法，其包括：在目的猪基因组的BMP15基因的外显子1上设计两条gRNA，分别将其构建至pX458载体和pX459载体，使所述BMP15基因的靶点切割区域的部分DNA片段被精确删除而终止表达，其中所述靶点切割区域的核苷酸序列如SEQ ID NO.6所示，其为如SEQ ID NO.1所示的核苷酸序列自5，末端第166-493位。
2.根据权利要求1所述的方法，其特征在于：被精确删除的所述部分DNA片段的核苷酸序列如SEQ ID NO.7所示，其为如SEQ ID NO.1所示的核苷酸序列自5，末端第299-470位。
3.根据权利要求1所述的方法，其特征在于：所述两条gRNA包括gRNA-E1S1和gRNA-E1S2，其均为单链DNA分子，gRNA-E1S1的核苷酸序列如SEQ ID NO.2所示，且gRNA-E1S2的核苷酸序列如SEQ ID NO.3所示。
4.根据权利要求3所述的方法，其特征在于：所述CRISPR/Cas9基因编辑包括如下步骤：
(1)将gRNA-E1S1构建到能表达Cas9蛋白和EGFP荧光蛋白的pX458载体上，得到pX458-gRNA-E1S1；将gRNA-E1S2构建到能表达Cas9蛋白和DsRed荧光蛋白的pX459载体上，得到pX459-gRNA-E1S2；将构建好的所述pX458-gRNA-E1S1和所述pX459-gRNA-E1S2共转染目的猪离体胎儿肾细胞中，得到BMP15基因编辑细胞群；
(2)通过用于扩增所述BMP15基因的外显子1的所述靶点切割区域的引物对对所述BMP15基因编辑细胞群进行PCR扩增，通过T-A克隆方法检测PCR扩增产物，计算克隆中含有编辑型BMP15基因的克隆比例，即为CRISPR/Cas9系统编辑效率。
5.根据权利要求4所述的方法，其特征在于：所述用于扩增所述靶点切割区域的引物对包括核苷酸序列如SEQ ID NO.4所示的单链DNA分子以及核苷酸序列如SEQ ID NO.5所示的单链DNA分子。
6.根据权利要求4所述的方法，其特征在于：所述编辑型BMP15基因为野生型BMP15基因外显子1中172bp大片段缺失使所述BMP15基因丧失功能所得到的基因型。
7.根据权利要求6所述的方法，其特征在于：所述野生型BMP15基因的核苷酸序列的核苷酸序列如SEQ ID NO.1所示。
8.根据权利要求1-7中任一项所述的方法，其特征在于：所述猪为大白猪。
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