CN109576267A - 一种用于单碱基编辑的gRNA、载体、细胞及其制备方法 - Google Patents
一种用于单碱基编辑的gRNA、载体、细胞及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于单碱基编辑的gRNA，其核苷酸序列如SEQ ID NO.1所示。本发明利用该gRNA，将IGF2基因相应位点的胞嘧啶突变为胸腺嘧啶，解除ZBED6对IGF2表达的抑制作用，提高产肉量。
Description
技术领域
本发明属于生物技术领域，具体涉及一种用于单碱基编辑的gRNA、载体、细胞及其构建方法，以及一种利用单碱基编辑技术提高动物产肉量的方法。
背景技术
IGF2(Insulin-like Growth Factor 2，胰岛素样生长因子2)作为最复杂多样的生长因子之一，对猪胚胎发育以及出生后生长发育和肌肉生成起着非常重要的作用。
研究表明，IGF2基因的上调表达能够提高猪的产肉量，并降低背膘厚。ZBED6是一种能够结合在IGF2基因内含子3的3068-3072区域(GCTCG)的抑制子，对IGF2基因的转录存在较强的抑制作用，在多数中国地方猪种中，ZBED6与IGF2基因内含子3的3068-3072(GCTCG)区域结合，抑制IGF2的表达，这是导致中国地方猪种瘦肉率不高的一个重要因素。
因此，亟需一种能够有效提高猪产肉量的方法。
发明内容
本发明的目的是针对以上要解决的技术问题，提供一种通过单碱基编辑技术编辑IGF2基因来提高中国地方猪种产肉量的方法。
为了实现以上发明目的，本发明提供了以下技术方案：
在一个方面，本发明提供了一种用于单碱基编辑的gRNA，其核苷酸序列如SEQ IDNO.1所示。
作为一种优选的实施方案，所述gRNA针对IGF2基因第三内含子的3068-3072区域。作为一种更优选的实施方案，在IGF2基因第三内含子的3068-3072区域内的3071位点发生胞嘧啶(C)突变为胸腺嘧啶(T)。
作为一种更优选的实施方案，所述IGF2基因是猪IGF2基因，所述猪包括但不限于两广小花猪。
在另一个方面，本发明提供了一种用于单碱基编辑的载体，其包括如上所述的gRNA、以及至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体。该gRNA的核苷酸序列如SEQ ID NO.1所示。作为一种更优选的实施方案，所述至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体还可以表达绿色荧光蛋白，即该表达载体可以同时表达dCas9蛋白、胞嘧啶脱氨酶和绿色荧光蛋白。作为一种更优选的实施方案，该表达载体可以是含有单碱基编辑器(例如BE1、BE2、BE3)的CRISPR/Cas9载体，包括但不限于pX458-BE3载体。作为一种更优选的实施方案，以pX458-BE3载体为例，将gRNA构建到同时表达dCas9蛋白、胞嘧啶脱氨酶和绿色荧光蛋白的pX458-BE3载体上，得到pX458-BE3-gRNA。所述pX458-BE3-gRNA的功能包括：gRNA识别并引导dCas9蛋白和胞嘧啶脱氨酶至IGF2基因目标区域，dCas9蛋白打开目标区域DNA双链，胞嘧啶脱氨酶将目标区域胞嘧啶突变(C)为胸腺嘧啶(T)。
在另一个方面，本发明提供了一种细胞，其含有上述用于单碱基编辑的载体，该用于单碱基编辑的载体包括核苷酸序列如SEQ ID NO.1所示的gRNA、以及至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体。作为一种优选的实施方案，所述至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体还可以表达绿色荧光蛋白，即该表达载体可以同时表达dCas9蛋白、胞嘧啶脱氨酶和绿色荧光蛋白。作为一种更优选的实施方案，该表达载体可以是含有单碱基编辑器(例如BE1、BE2、BE3)的CRISPR/Cas9载体，包括但不限于pX458-BE3载体。
在另一个方面，本发明提供了制备上述细胞的方法，其包括：
步骤1：设计并合成gRNA，所述gRNA的核苷酸序列如SEQ ID NO.1所示；
步骤2：将所述gRNA构建到至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体上，得到含有所述gRNA的载体；
步骤3：将含有所述gRNA的载体转染动物细胞。
作为一种优选的实施方案，所述动物为哺乳动物，包括但不限于猪，特别是两广小花猪。
作为一种优选的实施方案，所述步骤2中，所述至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体还可以表达绿色荧光蛋白，即该表达载体可以同时表达dCas9蛋白、胞嘧啶脱氨酶和绿色荧光蛋白。作为一种更优选的实施方案，该表达载体可以是含有单碱基编辑器(例如BE1、BE2、BE3)的CRISPR/Cas9载体，包括但不限于pX458-BE3载体。
作为一种更优选的实施方案，所述方法还包括：将含有所述gRNA的载体转染动物细胞之后，流式分选包含荧光报告基因信号的单细胞克隆。作为一种更优选的实施方案，利用载体携带的绿色荧光蛋白，流式分选包含荧光报告基因信号的单细胞克隆，将获得的单细胞克隆扩大培养后提取基因组，进行基因组水平的鉴定，筛选得到含有IGF2单碱基编辑的单细胞克隆。作为一种更优选的实施方案，用于基因组水平鉴定的引物对包括核苷酸序列如SEQ ID NO.2所示的单链DNA分子以及核苷酸序列如SEQ ID NO.3所示的单链DNA分子。
作为一种优选的实施方案，所述动物细胞是猪离体胎儿成纤维细胞。
在另一个方面，本发明提供了一种利用单碱基编辑技术提高动物产肉量的方法，其包括：利用核苷酸序列如SEQ ID NO.1所示的gRNA使IGF2基因(特别是猪IGF2基因)第三内含子的3068-3072区域(GCTCG)(该区域为ZBED6抑制子的结合区域，ZBED6的结合会抑制IGF2基因的转录表达。)内的3071位点发生胞嘧啶(C)突变为胸腺嘧啶(T)。作为一种更优选的实施方案，在目的猪基因组的IGF2基因的目标区域设计核苷酸序列如SEQ ID NO.1所示的gRNA，将其构建至pX458-BE3载体，在CRISPR/dCas9的引导下，利用胞嘧啶脱氨酶的作用，将IGF2基因相应位点的胞嘧啶突变为胸腺嘧啶，并且不引入DNA的双链断裂。
步骤1：设计并合成gRNA，所述gRNA的核苷酸序列如SEQ ID NO.1所示；
步骤2：将所述gRNA构建到至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体上，得到含有所述gRNA的载体；
步骤3：将含有所述gRNA的载体转染动物细胞，得到含有IGF2单碱基编辑的单细胞克隆；
步骤4：利用体细胞核移植技术，将所述含有IGF2单碱基编辑的单细胞克隆培养至对数生长期时进行体细胞核移植和胚胎移植，制备IGF2单碱基编辑动物。
作为一种优选的实施方案，所述步骤2中，所述至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体还可以表达绿色荧光蛋白，即该表达载体可以同时表达dCas9蛋白、胞嘧啶脱氨酶和绿色荧光蛋白。作为一种更优选的实施方案，该表达载体可以是含有单碱基编辑器(例如BE1、BE2、BE3)的CRISPR/Cas9载体，包括但不限于pX458-BE3载体。
作为一种更优选的实施方案，该利用单碱基编辑技术提高动物产肉量的方法还包括：将含有所述gRNA的载体转染动物细胞之后，流式分选包含荧光报告基因信号的单细胞克隆。
作为一种优选的实施方案，所述动物为哺乳动物，包括但不限于猪，特别是两广小花猪。
本发明的实验证明，本发明通过单碱基编辑技术对IGF2基因内含子3的ZBED6结合基序作精确的遗传修饰，使猪IGF2基因第三内含子的3068-3072区域内的3071位点发生C>T突变，解除了ZBED6对IGF2表达的抑制作用，得到高产肉量(瘦肉率)的IGF2单碱基编辑猪。传统瘦肉率选育需要大量的生长和屠宰性能测定，测定周期长，测定费用高，瘦肉率提高较为缓慢。本方法制备的IGF2单碱基编辑猪，在基因组中不引入任何其他的外源DNA序列，可以使本地猪种保持基因组的“纯度”，最大限度地保留其肉质优良、口感好等优良性状，又能特异地提高其瘦肉率，对于改良和利用本地猪种资源具有重要的育种意义。
附图说明
图1为pCMV-BE3载体的结构示意图。
图2为pX458质粒结构示意图。
图3为pX458-BE3质粒结构示意图。
图4为pX458-BE3-gRNA对猪IGF2基因目标区域进行单碱基编辑的示意图。
图5为T-A克隆测序分析单碱基编辑效率的结果。
图6为qPCR检测IGF2单碱基编辑对细胞的增殖与成肌分化等相关因子表达水平的影响的结果。
图7为所获得的IGF2单碱基编辑猪与野生型猪的体型对照图片。
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，均为本领域技术人员所熟知的常规方法及技术手段。
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。
本发明所采用的单碱基编辑技术针对的(例如猪)IGF2基因的目标区域为IGF2基因第三内含子的3068-3072区域(GCTCG)。
通过单碱基编辑技术对IGF2基因内含子3的ZBED6结合基序作精确的遗传修饰，使3071位点发生C>T突变，解除ZBED6对IGF2表达的抑制作用，上调IGF2的表达。
实施例1：
利用单碱基编辑技术编辑猪IGF2基因，制备相关基因编辑细胞
1、离体猪胎儿成纤维细胞的获得
两广小花猪胎儿成纤维细胞从广东小耳花猪胎儿中分离建立。用剪刀和镊子取下35日龄两广小花猪胚胎，将取下的组织依次在75％v/v酒精以及添加了抗生素(100U/mL青霉素，100μg/mL硫酸链霉素)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(100U/mL青霉素，100μg/mL硫酸链霉素)的含20％(V/V)FBS(胎牛血清)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，可观察到猪胎儿成纤维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、IGF2单碱基编辑细胞的获得
(1)转染pX458-BE3-gRNA，获得IGF2单碱基编辑混合细胞群
针对猪IGF2基因第三内含子的3068-3072区域(GCTCG)设计一条gRNA，将其构建至pX458-BE3表达载体，在CRISPR/dCas9的引导下，利用胞嘧啶脱氨酶的作用，将IGF2基因相应位点的胞嘧啶突变为胸腺嘧啶。(如图4所示)。
其中，所述pX458-BE3、pX458-BE3-gRNA表达载体的构建方法如下。
pX458-BE3的构建步骤：
将pCMV-BE3(见图1)载体上的BE3编码序列去掉终止密码子后(SEQ ID NO.4，即将pCMV-BE3载体上的BE3编码序列去掉终止密码子后的序列)，将该序列克隆进删除了3×FLAG-Cas9序列的pX458载体骨架(见图2)，获得目的载体pX458-BE3(见图3)。
pX458-BE3-gRNA的构建步骤：
设计用于编辑猪IGF2基因的gRNA序列如下：
gRNA：5-CTCGCAGCGCGGGAGCGCGT-3，(SEQ ID NO.1)；
合成相应的寡核苷酸正负单链，每条单链上添加有相应的黏性末端。用BbsⅠ酶切1μg pX458-BE3载体，线性化的质粒经电泳后，通过胶回试剂盒回收酶切载体。同时，对正负两条寡核苷酸链进行退火和磷酸化处理，使之形成具有黏性末端的双链DNA短片段。将上述短片段和线性化的pX458-BE3载体通过T4连接酶进行连接反应，接着转化至DH5α感受态细胞中。最后挑选细菌单克隆，通过测序鉴定pX458-BE3-gRNA表达载体是否构建成功。
采用电转的方法按10μg pX458-BE3-gRNA质粒转染1*106猪胎儿成纤维细胞。电转严格按照试剂盒和电转仪说明书操作。
(2)流式分选EGFP(增强绿色荧光蛋白)阳性细胞
pX458-BE3-gRNA转染进细胞后，表达EGFP绿色荧光，48h后通过流式分选出的绿色荧光细胞，即为携带pX458-BE3-gRNA载体的细胞。
(3)鉴定IGF2单碱基编辑混合细胞群
设计用于扩增IGF2目标区域的引物对如下：
IGF2-F：5-CTTTAAGGAACCAGGTTTTCGCAGC-3，(SEQ ID NO.2)；
IGF2-R：5-GTGCTTTGAGGTCTCTGGAAGTTAG-3，(SEQ ID NO.3)。
将上述步骤(2)得到的IGF2单碱基编辑混合细胞群基因组作为模板，用上述IGF2-F与IGF2-R组成的引物对进行PCR扩增。PCR反应条件为：95℃预变性5min；95℃变性30s,64℃退火30s,72℃延伸1min，共36个循环；72℃延伸10min。PCR产物连接T载体后进行进一步的测序分析，测序结果表明，单碱基编辑效率为8.89％(见图5)。
(4)qPCR检测IGF2单碱基编辑混合细胞群的IGF2以及IGF2相关因子的表达水平
qPCR结果显示，单碱基编辑显著提高了IGF2以及成肌关键因子MyoD1的表达水平，同时细胞周期蛋白Cyclin D1、抗凋亡因子Bcl-2和Bcl-xl以及肌管形成标记基因myogenin和desmin的表达水平也有相应提高。这些结果表明IGF2基因的单碱基编辑能够促进胎儿成纤维细胞的增殖，增强其成肌分化潜能(见图6)。
设计用于qPCR检测的引物对如下：
| 引物 | 目的基因 | 序列 |
| Primer_For | beta-actin | TGGGCTACACTGAGGACC |
| Primer_Rev | GTCAAGCTCATTTCCTGGTAC | |
| Primer_For | Igf2 | CCCCAGTGAGACTCTGTGCG |
| Primer_Rev | CCAGGTGTCATAGCGGAAGAA | |
| Primer_For | Zbed6 | AAATCTAAGCCTTGTATCCT |
| Primer_Rev | GCTGAACTATCAATAGACCC | |
| Primer_For | MyoD1 | CAAACGCAAGACCACTAACGC |
| Primer_Rev | AATCAGCGGCTGCCCAAG | |
| Primer_For | Myogenin | CAGTCCAGAATGGGGCAGT |
| Primer_Rev | CCAGGGTTCAAGAGGTCC | |
| Primer_For | desmin | TCCAAGCCAGACCTCACC |
| Primer_Rev | CAGGAAGCACCTCCAAACT | |
| Primer_For | Cyclin D1 | CAGATCATCCGCAAACACG |
| Primer_Rev | CGGGAGACAGCAGCAGAGT | |
| Primer_For | Bcl-2 | CCCCTGGTGGACAACATC |
| Primer_Rev | GCAGCCCAGACTCACATT | |
| Primer_For | Bcl-xl | AGCGTATCAGAGCTTTGAGC |
| Primer_Rev | AATACCTGCATCTCCTTGTCT |
(5)单细胞克隆培养
细胞转染48小时后(确保细胞状态良好)，进行单细胞分选；分选前准备96孔板若干个，每个96孔板每孔加入150μl预热的条件培养基(50％v/v新鲜全DMEM和50％v/v已使用全DMEM混合过滤)；分选后，放入细胞培养箱，三天后，每孔加入50μl全DMEM培养基，一周后显微镜下观察单细胞克隆情况，并作相应标记，更换培养基。单细胞克隆堆积生长状态下，需要进行胰酶消化，加入培养基继续培养，培养至一定数量后进行基因型鉴定，基因型鉴定为IGF2单碱基编辑的单细胞克隆进行冻存保种以及后续体细胞核移植。
实施例2：利用体细胞核移植技术构建IGF2单碱基编辑猪
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％wt卵泡液、FSH(卵泡刺激素)、LH(黄体生成素)、EGF(表皮生长因子)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％wt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IGF2单碱基编辑的单细胞克隆，于5％v/vCO2、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IGF2单碱基编辑的单细胞克隆进行体细胞核移植，并在24h之内进行胚胎移植，制备IGF2单碱基编辑猪。BMP15编辑猪体细胞核移植及生产统计如表1所示。
表1
| 移植胚胎数 | 受体数 | 怀孕数(％) | 窝仔数 | 活仔数 | 健仔数 | |
| 1 | 1363 | 5 | 2(40％) | 16 | 11 | 8 |
| 2 | 948 | 5 | 1(20％) | 6 | 4 | 3 |
| 合计 | 2311 | 10 | 3(30％) | 22 | 15 | 11 |
如图7所示，“编辑型”为通过体细胞核移植所获得的IGF2单碱基编辑两广小花猪(猪IGF2基因第三内含子的3068-3072区域内的3071位点发生C>T突变所得到的基因型)，“野生型”为同日龄野生型两广小花猪，可见IGF2单碱基编辑猪肌肉更为发达，臀部、背部以及肩胛部肌肉与野生型相比最为明显。
序列表
<110> 中山大学
<120> 一种用于单碱基编辑的gRNA、载体、细胞及其制备方法
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ctcgcagcgc gggagcgcgt 20
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctttaaggaa ccaggttttc gcagc 25
<210> 3
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gtgctttgag gtctctggaa gttag 25
<210> 4
<211> 5130
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60
catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120
gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180
cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 240
aggtgcagca ttacctggtt tctcagctgg agcccatgcg gcgaatgtag tagggccatc 300
actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360
caccacgctg acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact 420
atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480
ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540
gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600
ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660
cacattctct gggccaccgg gttgaaaagc ggcagcgaga ctcccgggac ctcagagtcc 720
gccacacccg aaagtgataa aaagtattct attggtttag ccatcggcac taattccgtt 780
ggatgggctg tcataaccga tgaatacaaa gtaccttcaa agaaatttaa ggtgttgggg 840
aacacagacc gtcattcgat taaaaagaat cttatcggtg ccctcctatt cgatagtggc 900
gaaacggcag aggcgactcg cctgaaacga accgctcgga gaaggtatac acgtcgcaag 960
aaccgaatat gttacttaca agaaattttt agcaatgaga tggccaaagt tgacgattct 1020
ttctttcacc gtttggaaga gtccttcctt gtcgaagagg acaagaaaca tgaacggcac 1080
cccatctttg gaaacatagt agatgaggtg gcatatcatg aaaagtaccc aacgatttat 1140
cacctcagaa aaaagctagt tgactcaact gataaagcgg acctgaggtt aatctacttg 1200
gctcttgccc atatgataaa gttccgtggg cactttctca ttgagggtga tctaaatccg 1260
gacaactcgg atgtcgacaa actgttcatc cagttagtac aaacctataa tcagttgttt 1320
gaagagaacc ctataaatgc aagtggcgtg gatgcgaagg ctattcttag cgcccgcctc 1380
tctaaatccc gacggctaga aaacctgatc gcacaattac ccggagagaa gaaaaatggg 1440
ttgttcggta accttatagc gctctcacta ggcctgacac caaattttaa gtcgaacttc 1500
gacttagctg aagatgccaa attgcagctt agtaaggaca cgtacgatga cgatctcgac 1560
aatctactgg cacaaattgg agatcagtat gcggacttat ttttggctgc caaaaacctt 1620
agcgatgcaa tcctcctatc tgacatactg agagttaata ctgagattac caaggcgccg 1680
ttatccgctt caatgatcaa aaggtacgat gaacatcacc aagacttgac acttctcaag 1740
gccctagtcc gtcagcaact gcctgagaaa tataaggaaa tattctttga tcagtcgaaa 1800
aacgggtacg caggttatat tgacggcgga gcgagtcaag aggaattcta caagtttatc 1860
aaacccatat tagagaagat ggatgggacg gaagagttgc ttgtaaaact caatcgcgaa 1920
gatctactgc gaaagcagcg gactttcgac aacggtagca ttccacatca aatccactta 1980
ggcgaattgc atgctatact tagaaggcag gaggattttt atccgttcct caaagacaat 2040
cgtgaaaaga ttgagaaaat cctaaccttt cgcatacctt actatgtggg acccctggcc 2100
cgagggaact ctcggttcgc atggatgaca agaaagtccg aagaaacgat tactccatgg 2160
aattttgagg aagttgtcga taaaggtgcg tcagctcaat cgttcatcga gaggatgacc 2220
aactttgaca agaatttacc gaacgaaaaa gtattgccta agcacagttt actttacgag 2280
tatttcacag tgtacaatga actcacgaaa gttaagtatg tcactgaggg catgcgtaaa 2340
cccgcctttc taagcggaga acagaagaaa gcaatagtag atctgttatt caagaccaac 2400
cgcaaagtga cagttaagca attgaaagag gactacttta agaaaattga atgcttcgat 2460
tctgtcgaga tctccggggt agaagatcga tttaatgcgt cacttggtac gtatcatgac 2520
ctcctaaaga taattaaaga taaggacttc ctggataacg aagagaatga agatatctta 2580
gaagatatag tgttgactct taccctcttt gaagatcggg aaatgattga ggaaagacta 2640
aaaacatacg ctcacctgtt cgacgataag gttatgaaac agttaaagag gcgtcgctat 2700
acgggctggg gacgattgtc gcggaaactt atcaacggga taagagacaa gcaaagtggt 2760
aaaactattc tcgattttct aaagagcgac ggcttcgcca ataggaactt tatgcagctg 2820
atccatgatg actctttaac cttcaaagag gatatacaaa aggcacaggt ttccggacaa 2880
ggggactcat tgcacgaaca tattgcgaat cttgctggtt cgccagccat caaaaagggc 2940
atactccaga cagtcaaagt agtggatgag ctagttaagg tcatgggacg tcacaaaccg 3000
gaaaacattg taatcgagat ggcacgcgaa aatcaaacga ctcagaaggg gcaaaaaaac 3060
agtcgagagc ggatgaagag aatagaagag ggtattaaag aactgggcag ccagatctta 3120
aaggagcatc ctgtggaaaa tacccaattg cagaacgaga aactttacct ctattaccta 3180
caaaatggaa gggacatgta tgttgatcag gaactggaca taaaccgttt atctgattac 3240
gacgtcgatc acattgtacc ccaatccttt ttgaaggacg attcaatcga caataaagtg 3300
cttacacgct cggataagaa ccgagggaaa agtgacaatg ttccaagcga ggaagtcgta 3360
aagaaaatga agaactattg gcggcagctc ctaaatgcga aactgataac gcaaagaaag 3420
ttcgataact taactaaagc tgagaggggt ggcttgtctg aacttgacaa ggccggattt 3480
attaaacgtc agctcgtgga aacccgccaa atcacaaagc atgttgcaca gatactagat 3540
tcccgaatga atacgaaata cgacgagaac gataagctga ttcgggaagt caaagtaatc 3600
actttaaagt caaaattggt gtcggacttc agaaaggatt ttcaattcta taaagttagg 3660
gagataaata actaccacca tgcgcacgac gcttatctta atgccgtcgt agggaccgca 3720
ctcattaaga aatacccgaa gctagaaagt gagtttgtgt atggtgatta caaagtttat 3780
gacgtccgta agatgatcgc gaaaagcgaa caggagatag gcaaggctac agccaaatac 3840
ttcttttatt ctaacattat gaatttcttt aagacggaaa tcactctggc aaacggagag 3900
atacgcaaac gacctttaat tgaaaccaat ggggagacag gtgaaatcgt atgggataag 3960
ggccgggact tcgcgacggt gagaaaagtt ttgtccatgc cccaagtcaa catagtaaag 4020
aaaactgagg tgcagaccgg agggttttca aaggaatcga ttcttccaaa aaggaatagt 4080
gataagctca tcgctcgtaa aaaggactgg gacccgaaaa agtacggtgg cttcgatagc 4140
cctacagttg cctattctgt cctagtagtg gcaaaagttg agaagggaaa atccaagaaa 4200
ctgaagtcag tcaaagaatt attggggata acgattatgg agcgctcgtc ttttgaaaag 4260
aaccccatcg acttccttga ggcgaaaggt tacaaggaag taaaaaagga tctcataatt 4320
aaactaccaa agtatagtct gtttgagtta gaaaatggcc gaaaacggat gttggctagc 4380
gccggagagc ttcaaaaggg gaacgaactc gcactaccgt ctaaatacgt gaatttcctg 4440
tatttagcgt cccattacga gaagttgaaa ggttcacctg aagataacga acagaagcaa 4500
ctttttgttg agcagcacaa acattatctc gacgaaatca tagagcaaat ttcggaattc 4560
agtaagagag tcatcctagc tgatgccaat ctggacaaag tattaagcgc atacaacaag 4620
cacagggata aacccatacg tgagcaggcg gaaaatatta tccatttgtt tactcttacc 4680
aacctcggcg ctccagccgc attcaagtat tttgacacaa cgatagatcg caaacgatac 4740
acttctacca aggaggtgct agacgcgaca ctgattcacc aatccatcac gggattatat 4800
gaaactcgga tagatttgtc acagcttggg ggtgactctg gtggttctac taatctgtca 4860
gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat cctcatgctc 4920
ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact cgtgcacacc 4980
gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc ccctgaatac 5040
aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa gatgctctct 5100
ggtggttctc ccaagaagaa gaggaaagtc 5130
<210> 5
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tgggctacac tgaggacc 18
<210> 6
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gtcaagctca tttcctggta c 21
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ccccagtgag actctgtgcg 20
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ccaggtgtca tagcggaaga a 21
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aaatctaagc cttgtatcct 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gctgaactat caatagaccc 20
<210> 11
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
caaacgcaag accactaacg c 21
<210> 12
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aatcagcggc tgcccaag 18
<210> 13
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cagtccagaa tggggcagt 19
<210> 14
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ccagggttca agaggtcc 18
<210> 15
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tccaagccag acctcacc 18
<210> 16
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
caggaagcac ctccaaact 19
<210> 17
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cagatcatcc gcaaacacg 19
<210> 18
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cgggagacag cagcagagt 19
<210> 19
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
cccctggtgg acaacatc 18
<210> 20
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gcagcccaga ctcacatt 18
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
agcgtatcag agctttgagc 20
<210> 22
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
aatacctgca tctccttgtc t 21
Claims (10)
1.一种用于单碱基编辑的gRNA，其核苷酸序列如SEQ ID NO.1所示。
2.一种用于单碱基编辑的载体，其包括如权利要求1所述的gRNA、以及至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体。
3.根据权利要求2所述的用于单碱基编辑的载体，其特征在于，所述至少同时表达dCas9蛋白和胞嘧啶脱氨酶的载体还表达绿色荧光蛋白。
4.一种细胞，其特征在于含有权利要求2或3所述的用于单碱基编辑的载体。
5.制备权利要求4所述细胞的方法，其包括：
步骤1：设计并合成gRNA，所述gRNA的核苷酸序列如SEQ ID NO.1所示；
步骤2：将所述gRNA构建到至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体上，得到含有所述gRNA的载体；
步骤3：将含有所述gRNA的载体转染动物细胞。
6.根据权利要求5所述的方法，其特征在于，所述步骤2中，所述至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体还表达绿色荧光蛋白。
7.根据权利要求6所述的方法，其特征在于，所述方法还包括：将含有所述gRNA的载体转染动物细胞之后，流式分选包含荧光报告基因信号的单细胞克隆。
8.一种利用单碱基编辑技术提高动物产肉量的方法，其包括：利用核苷酸序列如SEQID NO.1所示的gRNA使IGF2基因第三内含子的3068-3072区域内的3071位点发生胞嘧啶突变为胸腺嘧啶。
9.一种利用单碱基编辑技术提高动物产肉量的方法，其包括：
步骤1：设计并合成gRNA，所述gRNA的核苷酸序列如SEQ ID NO.1所示；
步骤2：将所述gRNA构建到至少同时表达dCas9蛋白和胞嘧啶脱氨酶的表达载体上，得到含有所述gRNA的载体；
步骤3：将含有所述gRNA的载体转染动物细胞，得到含有IGF2单碱基编辑的单细胞克隆；
步骤4：利用体细胞核移植技术，将所述含有IGF2单碱基编辑的单细胞克隆培养至对数生长期时进行体细胞核移植和胚胎移植，制备IGF2单碱基编辑动物。
10.根据权利要求9所述的方法，其特征在于，所述动物是猪。
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