CN108441519A - 在crispr/cas9基因编辑中提高同源修复效率的方法 - Google Patents
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Abstract
本发明提供一种在CRISPR/CAS9基因编辑中提高同源修复效率的新方法,其是利用CRISPR/CAS9系统进行基因编辑,通过将MTR值控制在1×105‑5×105的范围内,来提高同源修复效率。其中,所述MTR值为待敲入的外源基因DNA的摩尔数与待打靶基因摩尔数的比值。本发明依据细胞自身的同源修复机制和细胞周期特点,在基因编辑过程中通过增加修复模板DNA的用量,为基因准确修复提供丰富的模板,进而减少非同源末端连接的比例,使得修复的平衡更倾向于同源修复。本方法可使体外培养体细胞的基因修复效率提升到90%以上,比国际现行方法高出30%,具有广阔的应用前景。
Description
技术领域
本发明涉及基因编辑技术,具体地说,涉及一种在CRISPR/CAS9基因编辑中提高同源修复效率的方法。
背景技术
在细胞和活体水平上进行遗传缺陷修复,已经成为了疾病的细胞治疗、新药开发、动物表型改善的首要手段与方法。在任何动物的细胞治疗中,收集动物的活体细胞,在体外进行分离培养、利用基因编辑技术修正缺陷基因、质量控制后回输到动物体内,进而达到治疗某些疾病的目的。细胞治疗已经发展成现代医学的主要发展方向。动物基因编辑主要用于获得生产性能提高、无免疫排斥基因(实现人源化)、生物活性物质的大量产生(生物提取制药)等高附加值的产品,如生物制药中的动物生物反应器、用于异种移植治疗器官衰竭的人源化器官生产、抗烈性传染病的畜禽新品种培育等诸多领域。目前已经获得了白化的基因编辑五指山猪、细毛产量明显增加的基因编辑羊、产肉性能与国际著名肉牛品种相当的基因编辑我国著名的鲁西黄牛等,这些优良性状是常规育种往往需要20年的时间都难以实现的,充分展示了基因编辑技术的巨大优势。
在疾病基因修复或定点插入新基因过程中,如何进行高效修复与定点整合是制约治疗用细胞或基因编辑动物规模化生产的主要障碍。在目前基因编辑的研究中主要侧重于研究CRISPR/CAS9基因本身的功能与活性、编辑效率和脱靶性等方面,而修复领域的研究较少,完全由细胞本身的修复系统完成,人为介入较少,效率难于控制,进展缓慢。目前国际上利用CRISPR/CAS基因编辑技术的同源修复效率仅为30%-60%,难于获得同源修复纯合子,只有通过大量的细胞筛选方能得到纯合子,工作繁杂、任务重、人物力消耗大。因此高效同源修复已成为当前研究热点。
发明内容
本发明的目的是提供一种在CRISPR/CAS9基因编辑中提高同源修复效率的新方法,以解决细胞(含单细胞受精卵)中同源修复效率低,同源修复纯合子比例低,阳性细胞筛选难度大,基因编辑个体成功率低等问题。
本发明的构思如下:在不改变原有基于CRISPR/CAS9技术的基因编辑操作过程的基础上,仅改变其中一个成分的比例,即通过增加待修复基因的同源臂DNA的用量来提高同源修复效率,就可以达到提高同源修复效率之目的。
为了实现本发明目的,本发明提供的在CRISPR/CAS9基因编辑中提高同源修复效率的方法,其是利用CRISPR/CAS9系统进行基因编辑,通过将MTR值控制在1×105-5×105的范围,来提高同源修复效率。
本发明中,所述MTR值(MTR,Model DNA:Target Ratio)为待敲入的外源基因DNA的摩尔数与待打靶基因摩尔数的比值。
本发明中,所述待修复基因摩尔数的计算方法为:待转染细胞的总数×2×10-23/6.023。
本发明中,所述待转染细胞为体细胞(如胎儿成纤维细胞)或非人类胚胎细胞(如含单细胞的受精卵)。
本发明中,所述待转染细胞来自于鼠、猪、牛、羊、猪或猴等哺乳动物。
前述方法包括以下步骤:
1)同源臂构建体的制备:将待打靶基因的同源左臂、待敲入的外源基因与待打靶基因的同源右臂顺次连接,即为同源臂构建体;
2)CAS9mRNA的合成;
3)sgRNA的合成;
4)采用脂质体介导的方法将上述同源臂构建体、CAS9mRNA和sgRNA共同转染体细胞,或者采用显微注射的方法将上述同源臂构建体、CAS9mRNA和sgRNA共同显微注射至胚胎细胞(含单细胞的受精卵);
5)中靶细胞的鉴定。
前述的方法,步骤1)中同源左、右臂大小分别为50-1000bp,待敲入的外源基因大小为1kb-7kb。优选地,同源左、右臂为等长。
前述的方法,步骤4)中采用脂质体介导的方法将所述同源臂构建体、CAS9mRNA和sgRNA共同转染体细胞,其中CAS9mRNA和sgRNA的摩尔比为1:2。
前述的方法,步骤4)中采用显微注射的方法将CAS9mRNA和sgRNA共同显微注射至胚胎细胞,其中CAS9mRNA和sgRNA的摩尔比为1:2。
本发明紧紧围绕细胞自身的同源修复机制和细胞周期特点,在基因编辑过程中通过增加待修复基因的同源臂DNA(修复模板DNA)的用量,为基因准确修复提供丰富的模板,进而减少非同源末端连接的比例,使得修复的平衡更倾向于同源修复,这也是细胞修复过程中首选的修复机制,以维持遗传基因的稳定性。
与现有技术相比,本发明具有以下优点:
采用本发明方法可使体外培养体细胞的基因修复效率提升到90%以上,比国际现行方法高出30%;双等位基因同时修复效率提升到60%以上;如果利用早期胚胎显微注射法基因修复效率可达60%以上,双等位基因同时修复纯合子修复效率可达40%以上。完全可以满足基因编辑动物或转基因动物规模化生产的需求,用于疾病的细胞治疗和生命科学的理论与应用研究。本方法具有操作简便、成本低和效果明显等优点,具有广阔的应用前景。
附图说明
图1为本发明实施例1中同源修复效率随着修复模板DNA添加剂量的增加而提高;其中,2444bp片段为同源修复片段,1416bp片段为非同源修复的原始长度;泳道1-6为6种不同的MTR量(0.11pM、0.34pM、0.68pM、1.02pM、1.36pM和1.7pM),泳道7为阴性对照,泳道8为DNA Marker。内参基因β-globin用于评价DNA的质量和数量。
图2为本发明实施例1中显微注射的原核期胚胎发育至囊胚的同源修复效率。其中,红色荧光即为同源修复的基因编辑胚胎。
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。
实施例1在CRISPR/CAS9基因编辑中提高同源修复效率的方法
本实施例以肌肉生长抑制素(MSTN)基因的修复为例,提供一种在CRISPR/CAS9基因编辑中提高同源修复效率的方法。
1、MTR计算方法
以离体培养,用于体外转染的106个细胞为例,MSTN等位基因含量为0.34×10- 5pMol,如果MTR值为5×105,则修复模板DNA(同源臂构建体)为1.7pMol。如果修复模板DNA上含有载体冗余序列,则仅计算含有同源臂在内的DNA分子量,进而计算出所需添加的修复模板DNA的量(pg或pM)。
2、胎儿成纤维细胞的体外基因编辑与同源修复
2.1体外培养胎儿成纤维细胞
采集妊娠40天的崂山奶山羊、长白猪、晋南牛等胎儿,将整个组织剪碎,在DMEM+10%FBS中进行原代培养,传到第3代时,留出一部分进行体外转染,其余部分进行冷冻保存(抗冻剂为DMSO或甘油),用于后期的复苏。
2.2胎儿成纤维细胞的体外转染
在CAS9mRNA(SEQ ID NO:4)与MSTN sgRNA(SEQ ID NO:5)添加量为常规转染剂量(CAS9mRNA为0.2pM,sgRNA为0.4pM)的基础上,以两侧同源臂长度各为1000bp(同源左臂和同源由臂的DNA序列分别见SEQ ID NO:1和2),中间为1000bp无关序列(待敲入外源基因的核酸序列见SEQ ID NO:3)的DNA修复模板(同源臂构建体)为例(与CAS9mRNA和sgRNA共转染),修复模板DNA的添加剂量分别为每百万个细胞(0.11pM、0.34pM、0.68pM、1.02pM、1.36pM和1.7pM)。则同源修复效率分别为43.24%、56.91%、79.84%、91.22%、91.28%和93.47%,同源修复效率随着修复模板数量的增加而增加(图1)。与所来源的物种没有明显联系,各物种成纤维细胞的同源修复效率间没有明显差异。
目前世界范围内的修复模板的添加剂量均小于每百万个细胞0.34pM(MTR值为1×105以下),修复效率为30%-60%之间。
2.3小鼠单细胞原核胚胎显微注射法修复模板DNA添加剂量分析
(1)小鼠超数排卵
取2月龄,体重为20g左右的成年健康小鼠进行超数排卵,下午腹腔注射PMSG 5IU,48小时后注射5IU HCG,之后与雄鼠合笼,检栓,5-18小时收集受精卵,平均每只供体鼠可获得胚胎40-50枚。
(2)单细胞受精卵的原核注射
按常规的CAS9mRNA(SEQ ID NO:4)和MSTN sgRNA(SEQ ID NO:9)的注射剂量(CAS9mRNA为200ng/μl,sgRNA为10ng/μl)进行原核胚胎的显微注射,以两侧同源臂长度各为1000bp(同源左臂和同源由臂的DNA序列分别见SEQ ID NO:6和7)、中间为3460bp红色荧光蛋白的编码基因(SEQ ID NO:8)的同源臂构建体为例(与CAS9mRNA和sgRNA共同显微注射),修复模板DNA(同源臂构建体)的用量为1.7×10-6pMol。其红色荧光蛋白表达的细胞数达到了60%以上(图2),同源修复效率有了明显提升。
采用本发明方法在离体细胞水平中靶点上同源修复效率可提高20%-30%,增加三分之一左右,这意味着筛选细胞的工作量将降低三分之一,也意味着试剂消耗等也将降低三分之一,更重的是可有效加快实验进程。另外可使基因编辑转基因动物的生产效率提高近一倍,成功率达到60%,使转基因动物的效率提升一倍。以每只转基因羊30万元计算,每次增加一倍,意味着可减少成本至每只15万元,其经济效益巨大。
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,可以对之做一些修改或改进,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。
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<400> 4
atggactata aggaccatga cggagactac aaggatcatg atattgatta caaagacgat 60
gacgataaga tggccccaaa gaagaagagg aaggtcggta tccatggagt cccagcagcc 120
gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 180
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgacaggcat 240
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 300
accaggctga agaggaccgc caggaggagg tacaccagga ggaagaacag gatctgctat 360
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccataggctg 420
gaagagtcct tcctggtgga agaggataag aagcatgaga ggcatcccat cttcggcaac 480
atcgtggacg aggtggccta ccatgagaag taccccacca tctaccatct gaggaagaaa 540
ctggtggaca gcaccgacaa ggccgacctg aggctgatct atctggccct ggcccatatg 600
atcaagttca ggggccattt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 660
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 720
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca ggctgagcaa gagcaggagg 780
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg 840
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 900
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 960
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 1020
ctgagcgaca tcctgagggt gaacaccgag atcaccaagg cccccctgag cgcctctatg 1080
atcaagaggt acgacgagca tcatcaggac ctgaccctgc tgaaagctct cgtgaggcag 1140
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1200
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1260
aagatggacg gcaccgagga actgctcgtg aagctgaaca gggaggacct gctgaggaag 1320
cagaggacct tcgacaacgg cagcatcccc catcagatcc atctgggaga gctgcatgcc 1380
attctgagga ggcaggaaga tttttaccca ttcctgaagg acaacaggga aaagatcgag 1440
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcagg 1500
ttcgcctgga tgaccaggaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1560
gtggacaagg gcgcttccgc ccagagcttc atcgagagga tgaccaactt cgataagaac 1620
ctgcccaacg agaaggtgct gcccaagcat agcctgctgt acgagtactt caccgtgtat 1680
aacgagctga ccaaagtgaa atacgtgacc gagggaatga ggaagcccgc cttcctgagc 1740
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaacaggaa agtgaccgtg 1800
aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1860
ggcgtggaag ataggttcaa cgcctccctg ggcacatacc atgatctgct gaaaattatc 1920
aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1980
accctgacac tgtttgagga cagggagatg atcgaggaaa ggctgaaaac ctatgcccat 2040
ctgttcgacg acaaagtgat gaagcagctg aagaggagga ggtacaccgg ctggggcagg 2100
ctgagcagga agctgatcaa cggcatcagg gacaagcagt ccggcaagac aatcctggat 2160
ttcctgaagt ccgacggctt cgccaacagg aacttcatgc agctgatcca tgacgacagc 2220
ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcat 2280
gagcatattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2340
aaggtggtgg acgagctcgt gaaagtgatg ggcaggcata agcccgagaa catcgtgatc 2400
gaaatggcca gggagaacca gaccacccag aagggacaga agaacagccg cgagaggatg 2460
aagaggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acatcccgtg 2520
gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggagggat 2580
atgtacgtgg accaggaact ggacatcaac aggctgtccg actacgatgt ggaccatatc 2640
gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac caggagcgac 2700
aagaacaggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2760
tactggaggc agctgctgaa cgccaagctg attacccaga ggaagttcga caatctgacc 2820
aaggccgaga ggggcggcct gagcgaactg gataaggccg gcttcatcaa gaggcagctg 2880
gtggaaacca ggcagatcac aaagcatgtg gcacagatcc tggactccag gatgaacact 2940
aagtacgacg agaatgacaa gctgatcagg gaagtgaaag tgatcaccct gaagtccaag 3000
ctggtgtccg atttcaggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 3060
catcatgccc atgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3120
cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gaggaagatg 3180
atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3240
atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatcag gaagaggcct 3300
ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggcag ggattttgcc 3360
accgtgagga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3420
acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3480
aggaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3540
tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3600
gagctgctgg ggatcaccat catggaaagg agcagcttcg agaagaatcc catcgacttt 3660
ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3720
tccctgttcg agctggaaaa cggcaggaag aggatgctgg cctctgccgg cgaactgcag 3780
aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccat 3840
tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3900
cataagcatt acctggacga gatcatcgag cagatcagcg agttctccaa gagggtgatc 3960
ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcatag ggataagccc 4020
atcagggagc aggccgagaa tatcatccat ctgtttaccc tgaccaatct gggagcccct 4080
gccgccttca agtactttga caccaccatc gacaggaaga ggtacaccag caccaaagag 4140
gtgctggacg ccaccctgat ccatcagagc atcaccggcc tgtacgagac aaggatcgac 4200
ctgtctcagc tgggaggcga caagaggcct gctgctacta agaaagctgg tcaagctaag 4260
aaaaagaaat aa 4272
<210> 5
<211> 168
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 5
uaauacgacu cacuauaggg gacaucuuug uaggaguaca gcaaguuuua gagcuagaaa 60
uagcaaguua aaauaaggcu aguccguuau caacuugaaa aaguggcacc gagucggugc 120
cuagcauaac cccuuggggc cucuaaacgg gucuugaggg guuuuuug 168
<210> 6
<211> 1000
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgctgcatg caaattcttc tcttccttct gttccttctg ctgtggtcct gaggattgac 60
ccagaacctt gcacatgacg ggcaaaccct gttccaccaa gctccattca ctcccagttc 120
atcacttctt ataaacctct tctttgctcc tttaaggatt ctctttccac gcactataca 180
tacagccact gtcatcatgc taatttctct cttcccactg taatccatat agtgctcaga 240
aagagctact ttgctctgac tttaatatgc acatttcttt cagaaaagac ctaggggaga 300
ccagatgcgt acctatccat ccaagagcca ttagtcacct gcaatcaaca atgatgaaaa 360
gcgatgctca gtcctctcac cgacccgccc accttccctc tgtttcatct tatacatatt 420
tcccaggcat ctgttctgct attacgtgct attatgtctg ataatagtat gaaaagaaac 480
aaaactttaa aggacacaaa aggaaggacc acagggaatg cctgatgctg ttagagtctt 540
tagggccatg aaaggaaaaa tgaagtctag tgtatataaa aattccttaa ttctgcagtt 600
cttttaaaaa aaaagcgtaa aaattatgtg gttggtttgt ttgtttgttt gtttgttttt 660
ctaataatga tttttaaggt aggaaggatt tcaggctcta tttacataat tgttctttcc 720
ttttcacaca gaatcccttt ttagaagtca aggtgacaga cacacccaag aggtcccgga 780
gagactttgg gcttgactgc gatgagcact ccacggaatc ccggtgctgc cgctaccccc 840
tcacggtcga ttttgaagcc tttggatggg actggattat cgcacccaaa agatataagg 900
ccaattactg ctcaggagag tgtgaatttg tgtttttaca aaaatatccg catactcatc 960
ttgtgcacca agcaaacccc agaggctcag caggcccttg 1000
<210> 7
<211> 1000
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctgcactccg acaaaaatgt ctcccattaa tatgctatat tttaatggca aagaacaaat 60
aatatatggg aaaattccag ccatggtagt agaccgctgt gggtgctcat gagctttgca 120
ttaggttaga aatttcccaa gtcatggaag gtcttcccct caatttcgaa actgtgaatt 180
caagcaccac aggctgtagg ccttgagtat gctctagtaa cgtaagcaca agctacagtg 240
tatgaactaa aagagagaat agatgcaatg gttggcattc aaccaccaaa ataaaccata 300
ctataggatg ttgtatgatt tccagagttt ttgaaataga tggagatcaa attacattta 360
tgtccatata tgtatattac aactacaatc taggcaagga agtgagagca catcttgtgg 420
tctgctgagt taggagggta tgattaaaag gtaaagtctt atttcctaac agtttcactt 480
aatatttacg gaagaatcta tatgtagcct ttgtaaagtg taggattgtt atcatttaaa 540
aacatcatgt acacttatat ttgtattgta tacttggtaa gataaaattc cacaaagtag 600
gaatggggcc ttacatacac attgccattc ctattataat tggacaatcc accacggtgc 660
taatgcagtg ctgaatggct cctactggac ctctcgatag aacactctac aaagtacgag 720
tctctctctc ccttccaggt gcatctccac acacacagca ctaagtgttc aatgcatttt 780
ctttaaggaa agaagaatct ttttttctag aggtcaactt tcagtcaact ctagcacagc 840
gggagtgact gctgcatctt aaaaggcagc caaacagtat tcatttttta atctaaattt 900
caaaatcact gtctgccttt atcacatggc aattttgtgg taaaataatg gaaatgactg 960
gttctatcaa tattgtataa aagactctga aacaattaca 1000
<210> 8
<211> 3460
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gctagcgcta ccggactcag atctcgagct caagcttcga attctgcagt cgacattgat 60
tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 120
agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 180
gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 240
gacgtcaatg ggtggactat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 300
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 360
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 420
ctattaccat gggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc 480
tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg 540
gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag 600
gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc 660
gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc 720
gttgccttcg ccccgtgccc cgctccgcgc cgcctcgcgc cgcccgcccc ggctctgact 780
gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 840
gcgcttggtt taatgacggc tcgtttcttt tctgtggctg cgtgaaagcc ttaaagggct 900
ccgggagggc cctttgtgcg ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg 960
tggggagcgc cgcgtgcggc ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc 1020
ggggctttgt gcgctccgcg tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg 1080
tgcggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag 1140
cagggggtgt gggcgcggcg gtcgggctgt aacccccccc tgcacccccc tccccgagtt 1200
gctgagcacg gcccggcttc gggtgcgggg ctccgtgcgg ggcgtggcgc ggggctcgcc 1260
gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc 1320
ggggagggct cgggggaggg gcgcggcggc cccggagcgc cggcggctgt cgaggcgcgg 1380
cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt 1440
cccaaatctg gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg 1500
gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg 1560
cgccgccgtc cccttctcca tctccagcct cggggctgcc gcagggggac ggctgccttc 1620
gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct 1680
ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta 1740
ttgtgctgtc tcatcatttt ggcaaagaat tcgccaccat ggtgagcaag ggcgaggagg 1800
tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg ctccatgaac ggccacgagt 1860
tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga 1920
aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc cagttcatgt 1980
acggctccaa ggcgtacgtg aagcaccccg ccgacatccc cgattacaag aagctgtcct 2040
tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggt ctggtgaccg 2100
tgacccagga ctcctccctg caggacggca cgctgatcta caaggtgaag atgcgcggca 2160
ccaacttccc ccccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcca 2220
ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccaccag gccctgaagc 2280
tgaaggacgg cggccactac ctggtggagt tcaagaccat ctacatggcc aagaagcccg 2340
tgcaactgcc cggctactac tacgtggaca ccaagctgga catcacctcc cacaacgagg 2400
actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg ttcctggggc 2460
atggcaccgg cagcaccggc agcggcagct ccggcaccgc ctcctccgag gacaacaaca 2520
tggccgtcat caaagagttc atgcgcttca aggtgcgcat ggagggctcc atgaacggcc 2580
acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca 2640
agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tccccccagt 2700
tcatgtacgg ctccaaggcg tacgtgaagc accccgccga catccccgat tacaagaagc 2760
tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggtctgg 2820
tgaccgtgac ccaggactcc tccctgcagg acggcacgct gatctacaag gtgaagatgc 2880
gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg ggctgggagg 2940
cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc caccaggccc 3000
tgaagctgaa ggacggcggc cactacctgg tggagttcaa gaccatctac atggccaaga 3060
agcccgtgca actgcccggc tactactacg tggacaccaa gctggacatc acctcccaca 3120
acgaggacta caccatcgtg gaacagtacg agcgctccga gggccgccac cacctgttcc 3180
tgtacggcat ggacgagctg tacaagtaat agcggccgcg actctagatc ataatcagcc 3240
ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc 3300
tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt 3360
acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 3420
gttgtggttt gtccaaactc atcaatgtat cttaagcacc 3460
<210> 9
<211> 168
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 9
uaauacgacu cacuauaggg gacauuuuug ucggagugca gcaaguuuua gagcuagaaa 60
uagcaaguua aaauaaggcu aguccguuau caacuugaaa aaguggcacc gagucggugc 120
cuagcauaac cccuuggggc cucuaaacgg gucuugaggg guuuuuug 168
Claims (9)
1.在CRISPR/CAS9基因编辑中提高同源修复效率的方法,其特征在于,利用CRISPR/CAS9系统进行基因编辑,通过将MTR值控制在1×105-5×105的范围,来提高同源修复效率;
其中,所述MTR值为待敲入的外源基因DNA的摩尔数与待打靶基因摩尔数的比值。
2.根据权利要求1所述的方法,其特征在于,所述待打靶基因摩尔数的计算方法为:待转染细胞的总数×2×10-23/6.023。
3.根据权利要求2所述的方法,其特征在于,所述待转染细胞为体细胞或非人类胚胎细胞。
4.根据权利要求3所述的方法,其特征在于,包括以下步骤:
1)同源臂构建体的制备:将待打靶基因的同源左臂、待敲入的外源基因与待打靶基因的同源右臂顺次连接,即为同源臂构建体;
2)CAS9 mRNA的合成;
3)sgRNA的合成;
4)采用脂质体介导的方法将上述同源臂构建体、CAS9 mRNA和sgRNA共同转染体细胞,或者采用显微注射的方法将上述同源臂构建体、CAS9 mRNA和sgRNA共同显微注射至胚胎细胞;
5)中靶细胞的鉴定。
5.根据权利要求4所述的方法,其特征在于,步骤1)中同源左、右臂大小分别为50-1000bp,待敲入的外源基因大小为1kb-7kb。
6.根据权利要求5所述的方法,其特征在于,步骤1)中同源左、右臂为等长。
7.根据权利要求4所述的方法,其特征在于,步骤4)中采用脂质体介导的方法将所述同源臂构建体、CAS9 mRNA和sgRNA共同转染体细胞,其中CAS9 mRNA和sgRNA的摩尔比为1:2。
8.根据权利要求4所述的方法,其特征在于,步骤4)中采用显微注射的方法将CAS9mRNA和sgRNA共同显微注射至胚胎细胞,其中CAS9 mRNA和sgRNA的摩尔比为1:2。
9.根据权利要求2所述的方法,其特征在于,所述待转染细胞来自于鼠、猪、牛、羊、猪或猴。
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