CN105950626B - 基于CRISPR/Cas9获得不同毛色绵羊的方法及靶向ASIP基因的sgRNA - Google Patents
基于CRISPR/Cas9获得不同毛色绵羊的方法及靶向ASIP基因的sgRNA Download PDFInfo
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Abstract
本发明公开了一种基于CRISPR/Cas9获得不同毛色绵羊的方法及靶向ASIP基因的sgRNA。本发明提供了一种能够特异的靶向修饰绵羊ASIP基因的sgRNA(ASIP‑sgRNA),为序列表的序列4自5’末端第3至22位核苷酸所示的RNA或具有序列表的序列4自5’末端第3至22位核苷酸的RNA。本发明的实施例中,所述ASIP‑sgRNA具体可为序列表的序列4所示的RNA。本发明还保护一种获得毛色改变的绵羊的方法,包括如下步骤:将所述能够特异的靶向修饰绵羊ASIP基因的sgRNA和Cas9mRNA共转染绵羊细胞,从而敲除绵羊ASIP基因,得到毛色改变的绵羊。本发明将新的CRISPR/Cas9基因组编辑技术与显微注射技术相结合,为人为改变绵羊毛色提供了有效的技术手段。
Description
技术领域
本发明属于动物基因工程领域,涉及CRISPR/Cas9技术,具体涉及一种基于CRISPR/Cas9基因敲除技术获得不同毛色绵羊的方法以及用于特异性靶向ASIP基因修饰的sgRNA。
背景技术
基因组操作技术是近年基于基因组和基因信息技术发展起来的通过人工设计实现对特定基因或基因组靶位点进行精确编辑的前沿技术,目前已经成为生物医学、农业动物育种和模式动物等领域的研究热点。在动物育种领域,要培育一个品种或建立一个专门化品系,必须以提高其生产性能,稳定其遗传基础为前提。在羊的育种工作中,毛色性状是一种重要的品种特征和生产性状,在确定杂交组合、品种纯度和亲缘关系等方面意义重大,尤其在通过杂交育种获得所需要的毛色类型上作用突出。因此,利用高效稳定的基因组操作技术来改变绵羊毛色对绵羊毛色选育工作至关重要。
近年来,科学家们根据细菌获得性免疫的原理,发明了基于CRISPR/Cas9的基因组编辑新技术,不仅大大降低了对动物进行基因敲除、基因修饰的难度,更是将动物转基因技术由传统的随机整合推向了高度精确的基因组定向删除、插入或替换等精准修饰,开创了转基因动物生产的新时代。CRISPR/Cas9系统是一个由核酸和蛋白质组成的核糖核蛋白复合物,它对靶点的识别依赖于核酸对核酸的识别,通过碱基的互补配对完成。打靶一个位点只需要根据目标序列设计合成20bp左右的寡核苷酸。这种基于碱基互补配对原则的识别,相比较于蛋白质与DNA之间的相互作用要更加稳定和简单,能够实现一次操作同时突变两个以上基因或位点,大大提高了基因组编辑技术效率。
由于CRISPR/Cas9系统中发挥活性作用的核心部件为sgRNA和蛋白质,因此可以通过构建载体,体外转录获得RNA后,显微注射动物受精卵而获得打靶动物,在整个打靶过程中不存在外源DNA的整合。而且由于mRNA的不稳定性,不会长期存在生物体内,也不会对环境产生进一步影响,因而可以避免传统转基因导致的生物安全问题。也就是说,CRISPR/Cas9技术修饰的是一个物种自身的基因,采取mRNA或RNA作为基因打靶原材料,没有任何筛选用抗性基因,因而不存在生物安全性问题。而且,获得的最终产品是经过了遗传修饰的(通过转基因的方法定点修饰,因为这个系统是细菌里的,所以要靠转基因的方法转到动植物中),但终产品里却可以不包含任何转基因的成分,大大提高了安全性。所以,在目前动植物新品种培育,尤其是转基因动物的制备中,CRISPR/Cas9介导的打靶系统被研究者广为采用。
ASIP(agouti signalling protein,野灰位点信号蛋白)基因是由agouti位点编码的一种信号蛋白,是影响绵羊毛色的主要候选基因,在色素形成中起重要的调控作用,它与α-促黑色素细胞激素(α-MSH)竞争结合黑素皮质激素受体1(melanocortin-1,MC1R),使MC1R结构改变,抑制环磷酸腺苷(cAMP)酶系统,引起cAMP水平下降,通过级联反应,促进褐黑素的产生。在绵羊中,ASIP基因被定位在13号染色体上,该基因在绵羊基因组中存在多个拷贝和多个等位基因,其编码序列主要由3个外显子、2个内含子和部分3’、5’非翻译区组成。绵羊Agouti基因编码的ASIP蛋白由133个氨基酸构成,由一段信号肽和功能氨基酸组成。大量研究已经证实,ASIP基因与绵羊的被毛颜色存在密切的相关性。Norris和Whan揭示了绵羊的白色被毛与ASIP基因组中190kbp的重复有关,而绵羊的黑色被毛则于第二外显子上的缺失和第四外显子上的错义突变有关。此外,不同绵羊品种由于ASIP基因拷贝数的变异和错义突变将会导致不同类型毛色的出现。
由于细毛羊经长期选育均为白色个体,通过新型基因组编辑技术敲除ASIP基因从而改变细毛羊毛色,对培育彩色细毛羊新品种或新类型、深入研究该基因遗传的分子基础,指导绵羊品种改良、选育都有重要用途。
发明内容
本发明的目的是提供一种基于CRISPR/Cas9基因敲除技术获得不同毛色绵羊的方法以及用于特异性靶向ASIP基因修饰的sgRNA。
本发明提供了一种能够特异的靶向修饰绵羊ASIP基因的sgRNA(ASIP-sgRNA),为序列表的序列4自5’末端第3至22位核苷酸所示的RNA或具有序列表的序列4自5’末端第3至22位核苷酸的RNA。本发明的实施例中,所述ASIP-sgRNA具体可为序列表的序列4所示的RNA。
本发明还保护编码所述ASIP-sgRNA的DNA分子。编码所述ASIP-sgRNA的DNA分子具体可为序列表的序列3所示的DNA分子。
本发明还保护一种能够特异的靶向修饰绵羊ASIP基因的靶向序列,为序列表的序列1自5’末端第359至378位核苷酸。
本发明还保护一种特异性敲除绵羊ASIP基因的成套核酸分子,包括所述的sgRNA。所述成套核酸分子还可包括Cas9mRNA。所述Cas9mRNA为编码序列表的序列6所示Cas9蛋白的RNA。所述Cas9mRNA具体可为具有序列表的序列7自5’末端第7至4278位核苷酸的RNA,更具体可为序列表的序列7所示的RNA。
本发明还保护一种特异性敲除绵羊ASIP基因的成套核酸分子,包括所述的DNA分子。所述成套脱氧核糖核酸分子还可包括编码Cas9mRNA的DNA分子。所述Cas9mRNA为编码序列表的序列6所示Cas9蛋白的RNA。所述Cas9mRNA具体可为具有序列表的序列7自5’末端第7至4278位核苷酸的RNA,更具体可为序列表的序列7所示的RNA。编码Cas9mRNA的DNA分子具体可为具有序列表的序列5自5’末端第24至4295位核苷酸的DNA分子,更具体可为序列表的序列5所示的分子。
本发明还保护一种特异性敲除绵羊ASIP基因的方法,包括如下步骤:将所述能够特异的靶向修饰绵羊ASIP基因的sgRNA和Cas9mRNA共转染绵羊细胞,从而敲除绵羊ASIP基因。所述Cas9mRNA为编码序列表的序列6所示Cas9蛋白的RNA。所述Cas9mRNA具体可为具有序列表的序列7自5’末端第7至4278位核苷酸的RNA,更具体可为序列表的序列7所示的RNA。所述共转染的方式具体可为共注射。所述绵羊细胞具体可为绵羊受精卵单细胞。
本发明还保护一种获得毛色改变的绵羊的方法,包括如下步骤:将所述能够特异的靶向修饰绵羊ASIP基因的sgRNA和Cas9mRNA共转染绵羊细胞,从而敲除绵羊ASIP基因,得到毛色改变的绵羊。
所述毛色改变的绵羊具体如下:毛色为黑色的绵羊、毛色为黑白花(黑/白斑点花纹)的绵羊或毛色为棕白花(棕色/白色花纹)的绵羊。
以上任一所述绵羊ASIP基因可为编码序列表的序列2所示的蛋白质的基因。以上任一所述绵羊ASIP基因具体可为编码区如序列表的序列8所示的DNA分子或序列表的序列1所示的DNA分子或序列表的序列1自5’末端第222至1398位核苷酸所示的DNA分子或如GENBANK ACCESSION NO.NC_019470中所示(VERSION NC_019470.1;GI:417531912;linearCON 03-APR-2015)的DNA分子。
以上任一所述绵羊具体可为新疆细毛羊。
目前在大动物基因组上实现修饰和改造的成本和技术要求仍然很高,因此获得特异、高效的sgRNA成为绵羊基因组编辑培育的关键。本发明提供的sgRNA特异性高且能够精确靶向修饰绵羊ASIP基因,实现基因突变。
本发明中,首次采用CRISPR/Cas9技术在绵羊受精卵中实现ASIP基因的精准修饰突变并获得不同毛色的基因编辑绵羊,利用这种方法不仅构建步骤简单,安全性高,而且大大降低了昂贵的实验成本和缩短实验周期,实现了绵羊毛色基因的精准编辑,获得了黑色、黑/白斑点花纹和棕色/白色花纹细毛羊。
本发明将新的CRISPR/Cas9基因组编辑技术与显微注射技术相结合,为人为改变绵羊毛色提供了有效的技术手段。
附图说明
图1为限制性内切酶BbsI单酶切px330质粒后的1%琼脂糖凝胶电泳图;泳道M为1kbDNA Marker。
图2为体外转录产物ASIP-sgRNA的凝胶电泳图;泳道M为RNA Marker,泳道1为sgRNAASIP。
图3为采用Cas9-F和Cas9-R组成的引物对进行PCR扩增的扩增产物电泳图;泳道M为1kb DNA Marker。
图4为Cas9mRNA的凝胶电泳图;泳道M为RNA Marker。
图5为绵羊胚胎PCR产物进行T7EN1酶切后的电泳图;1-19为试验处理组,Con1-5为对照处理组,泳道M为100bp DNA Marker。
图6为绵羊胚胎PCR产物测序结果;野生型(WT)中,标注红色碱基序列(下划直线)为sgRNA的靶序列,绿色碱基序列TGG(下划波浪线)为ASIP基因Cas9打靶的PAM序列;“^”下方红色碱基表示插入碱基,“-”表示删除碱基,突变碱基(方框)用红色表示。
图7为注射ASIP-Cas9mRNA后出生的不同毛色羔羊照片。
图8为羔羊ASIP基因PCR产物的电泳图;Con1-3代表非注射组生的羔羊,泳道M为100bp DNA Marker。
图9为突变羔羊的编辑形式;野生型(WT)中,标注红色碱基序列(下划直线)为sgRNA的靶序列,绿色碱基序列TGG(下划波浪线)为ASIP基因Cas9打靶的PAM序列;“^”下方红色碱基表示插入碱基,“-”表示删除碱基,突变碱基(方框)用蓝色表示。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明,此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。下述实施例中的实验方法,如无特殊说明,均为常规方法,详见《分子克隆(第三版)》。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂公司购买得到的。px330质粒:Addgene公司,货号为42330。RNA纯化试剂盒:Life Technologies公司,货号为AM1908。
实施例中的相关引物的核苷酸序列见表1。
表1 ASIP基因相关引物的核苷酸序列
引物 | 序列(5’to 3’) |
ASIP-CF | caccgTTTCCCTTCTGTCTCTATCG |
ASIP-CR | aaacCGATAGAGACAGAAGGGAAAc |
ASIP-TF | ttaatacgactcactataggTTTCCCTTCTGTCTCTATCG |
ASIP-TR | aaaagcaccgactcggtgcc |
ASIP-S | CCAAGGAAACAAAGAAAGCAG |
ASIP-AS | AACCAAACAAGTTAAGGGACA |
ASIP-in-S | CCTTCTCTGTCGCTCTCAAGCCTCC |
ASIP-in-AS | CTGAGGAATGAGCACAAAGGA |
注:各引物均为单链DNA分子。
实施例1、制备sgRNA和Cas9mRNA
一、设计绵羊ASIP基因靶序列以及识别靶序列的sgRNA
绵羊ASIP基因全长序列如GENBANK ACCESSION NO.NC_019470中所示(VERSIONNC_019470.1;GI:417531912;linear CON 03-APR-2015)。
序列表的序列1为绵羊ASIP基因的部分区段,其中具有三个外显子(第222-381位核苷酸、第795-859位核苷酸、第1222-1398位核苷酸),编码序列表的序列2所示的蛋白质。
经过大量预试验和验证试验,选择序列表的序列1中第359-378位核苷酸作为sgRNA针对绵羊ASIP基因的靶序列。
二、制备sgRNAASIP
1、用限制性内切酶BbsI酶单酶切px330质粒,然后进行1%琼脂糖凝胶电泳(酶切产物的1%琼脂糖凝胶电泳图见图1),切胶回收并纯化线性化的质粒。
2、将步骤1的纯化产物进行去磷酸化。
3、将引物ASIP-CF和引物ASIP-CR退火,得到两端均为粘末端的双链DNA分子。
4、将步骤2的产物与步骤3得到的双链DNA分子连接,得到重组质粒。
5、以步骤4得到的重组质粒为模板,采用ASIP-TF和ASIP-TR组成的引物对进行PCR扩增,得到PCR扩增产物。经测序,PCR扩增产物如序列表的序列3所示。
6、取步骤5得到的PCR扩增产物,利用体外转录试剂盒(T7Kit,Life Technologies公司,货号为AM1354)进行体外转录,然后采用RNA纯化试剂盒纯化回收,得到ASIP-sgRNA。ASIP-sgRNA的凝胶电泳图见图2。ASIP-sgRNA如序列表的序列4所示。
三、制备Cas9mRNA
1、以px330质粒为模板,采用Cas9-F(下划线标注T7启动子)和Cas9-R组成的引物对进行PCR扩增,得到PCR扩增产物(4311bp)。PCR扩增产物的电泳图见图3。经测序,PCR扩增产物如序列表的序列5所示。序列表的序列5中,自5’末端第24-4295位核苷酸为Cas9的开放阅读框。编码序列表的序列6所示的Cas9蛋白。
Cas9-F:5’-TAATACGACTCACTATAGGGAGAATGGACTATAAGGACCACGAC-3’;
Cas9-R:5’-GCGAGCTCTAGGAATTCTTAC-3’。
2、取步骤1得到的PCR扩增产物,利用体外转录试剂盒(Life Technologies公司的T7Ultra Kit,货号为AM1345)进行体外转录,然后采用RNA纯化试剂盒纯化回收,得到Cas9mRNA。Cas9mRNA的凝胶电泳图见图4。Cas9mRNA如序列表的序列7所示。Cas9mRNA自5’末端第7至4278位核苷酸为编码区。
实施例2、注射sgRNA/Cas9mRNA胚胎突变效率检测
一、绵羊受精卵的获得
1、卵母细胞的成熟
从屠宰场采集绵羊卵巢(卵巢来自哈萨克羊),用生理盐水灭菌清洗3-4次,抽取卵母细胞,用成熟液洗涤3-4次,然后滴入平衡好的成熟液滴(成熟液滴的体积为75-78μl,每滴滴入25-30枚卵母细胞),放入含5%CO2的38.6℃培养箱中培养(以下培养箱培养均为该相同条件)。
平衡成熟液:将成熟液滴在培养箱中放置2h。成熟液:TCM199培养液+体积百分含量为10%的FBS+0.05IU/ml FSH+0.05IU/ml LH+1μg/ml estradiol+24.2μg/ml丙酮酸钠+0.1mM/L半胱氨酸+10ng/ml EGF+100IU/ml青霉素+100IU/ml链霉素。
2、卵母细胞的体外受精
(1)将体外成熟24-26h的卵母细胞取出,用0.1%透明质酸酶轻轻吹打以除去颗粒细胞,再用受精液洗涤3次,然后放入平衡好的受精液滴内(每个受精液滴为50-70μl体外受精液,放入20-30枚卵母细胞)。
平衡受精液:将受精液滴在培养箱中放置3-4h。受精液:SOF液+体积百分含量为20%发情羊血清+6IU/ml肝素钠+100IU/ml庆大霉素。SOF液:含6.29mg/ml NaCl、0.534mg/ml KCl、0.162mg/ml KH2PO4、0.6μl/ml乳酸钠、0.089mg/ml MgSO4、2.1mg/ml NaHCO3、0.0357mg/ml丙酮酸钠和0.299mg/ml CaCL2·2H2O,溶剂为水。
(2)取冷冻的精液(精液来自哈萨克羊),水浴解冻,移入平衡好的受精液中,放入培养箱中20-25min(有活力的精子会向上游),然后吸取上部的精液,以1500rpm的转速离心4-5min,弃去上清液,得到精子沉淀(进行精子计数)。
(3)将步骤(2)得到的精子加入完成步骤(1)的受精液滴中,使精子的浓度为(2-4)×106个/ml,38.6℃静置孵育12-18h,用平衡好的培养液反复吹吸受精卵,然后按50-70枚/孔的密度移入四孔培养板内。
平衡好的培养液的制备方法:将培养液在培养箱中,放置3-4h。培养液:SOF液+3mg/ml BSA。
二、单细胞期受精卵的显微注射
1、将实施例1制备的ASIP-sgRNA和Cas9mRNA混合(用Nuclease-free Water调整终浓度分别为50ng/μL和100ng/μL)。
2、试验处理:采用NIKON公司的显微注射仪将步骤1得到的混合液注射入步骤一得到的处于单细胞期的受精卵的胞质内(每个受精卵注射80-100pL混合液),置于培养箱中培养。对照处理:采用NIKON公司的显微注射仪将Nuclease-free Water注射入步骤一得到的处于单细胞期的受精卵胞质内,置于培养箱中培养。
3、胚胎中靶基因编辑效率检测
(1)胚胎样品收集
步骤2中培养7d后,取胚胎,用PBS缓冲液洗涤2遍,然后置于5μL裂解液中,瞬时离心后37℃孵育3h。裂解液:溶剂为Tris-HCl(50mM、pH8.0),含0.5%(V/V)Triton X-100和1mg/mL Proteinase K。
(2)PCR扩增
以步骤(1)的裂解产物为模板进行巢式PCR。第一轮PCR扩增采用ASIP-S和ASIP-AS组成的引物对(PCR扩增片段长度为426bp),第二轮PCR扩增采用ASIP-in-S和ASIP-in-AS组成的引物对(PCR扩增片段长度为322bp)。
(3)T7核酸内切酶(T7EN1)及测序鉴定
分别将步骤(2)得到的对照组和试验组PCR产物等量混合,进行变性退火,形成异源杂交双链。退火程序:95℃10min;85℃、75℃、65℃、55℃、45℃、35℃、25℃各1min(降温速率0.3℃/s);10℃Pause。
在退火产物中加入T7EN1酶(NEB),37℃孵育30min,酶切产物进行2%的琼脂糖凝胶电泳。
T7EN1酶切结果见图5,试验组中发生突变的样品出现两种片段(大约183bp和139bp),而试验组中未发生突变的样品和对照组仅一种片段(322bp)。
将上述步骤(2)得到的PCR扩增产物进行测序。部分PCR扩增产物的测序比对结果如图6所示。发生ASIP基因突变的位置均毗邻PAM序列,突变类型包括2-10bp碱基的删除、2-8bp碱基的插入及碱基替换等形式。
全部PCR扩增产物的测序结果表明:绵羊胚胎发生ASIP基因删除/插入突变效率为55.96%(61/109),且基因敲除对绵羊胚胎发育无致死性,证明DNA分子被Cas9和sgRNA特异性编辑。
实施例3、基因编辑羊的生产
1、实验羊选择
选取体况优良、无繁殖疾病且在2-4岁的新疆细毛羊做供体母羊。选取体重在50kg以上,年龄为2-4岁,膘情好、无繁殖疾病的阿勒泰羊做受体母羊。选取体重在70-85kg,精液检测优良且在1-3岁的新疆细毛羊做采精公羊。
2、同期发情与超数排卵
绵羊发情周期内,供体母羊阴道放入CIDR阴道栓,放入CIDR阴道栓的第10天开始以递减的方式连续注射FSH(中国宁波三生公司),每隔12h注射一次,共3天,总剂量为240单位/只,在第12天早上取出CIDR栓,清洗阴道,并肌肉注射PG 0.1mg(中国宁波三生公司)。撤栓12h后开始用公羊试情,早晚各试情一次,间隔12h,供体母羊发情时注射LH 200IU/只(中国宁波三生公司)。
受体母羊与供体母羊同步埋植CIDR,在供体母羊撤栓前12h撤除CIDR,每只注射330IU PMSG(中国宁波三生公司),撤栓12h后每天早晚各2次用试情公羊试情,详细记录发情时间。
3、人工授精
人工采精公羊的精液,镜检,活率达0.8以上方可用于输精。对发情12-19h的供体母羊进行人工授精。
4、原核胚的获得
供体母羊输精后19-21h,手术法从输卵管中冲取原核胚。挑选出胞质均匀、形态规则、完整且致密的未卵裂的原核胚(单细胞期)。
5、将实施例1制备的ASIP-sgRNA和Cas9mRNA混合(用Nuclease-free Water调整终浓度分别为50ng/μL和100ng/μL)。
6、试验处理:采用NIKON公司的显微注射仪将步骤5得到的混合液注射入步骤4得到的处于单细胞期的受精卵的胞质内(每个受精卵注射80-100pL混合液),置于培养箱中培养。非注射组用等体积Nuclease-free Water代替所述混合液。
7、胚胎移植及妊娠检测
原核胚卵裂至2-4细胞时,将胚胎移植到同期发情处理的受体母羊的输卵管中,每侧输卵管移植1-2枚胚胎,移植60天后对受体母羊进行B超妊娠诊断。
8、靶向删除ASIP基因羊的鉴定
试验处理组受体母羊生产后得到6只羔羊,分别命名为GM081(毛色为黑色)、GM105(毛色为黑白花)、GM106(毛色为黑色)、GM108(毛色为白色)、GM109(毛色为黑白花)、GM110(毛色为棕白花)。羔羊出生后发现性状表现明显,大致呈现四种不同毛色:黑色、黑白色、棕白色和白色,部分见图7。
非注射组受体母羊生产后得到6只羔羊,均为白色。
(1)DNA提取及PCR扩增
出生后一周左右,采集试验处理组羔羊耳组织样品(同时采集同时期出生的非注射组羔羊耳组织做对照),提取基因组DNA,用ASIP-S和ASIP-AS组成的引物对进行PCR扩增(扩增的PCR产物片段长度为426bp)。PCR产物的电泳图见图8。
(2)TA克隆测序比对
将上述羔羊耳组织PCR产物分别克隆至pMD-19T载体,随机挑取20-30个单克隆测序来精确定位突变位点。发生突变的羔羊的编辑形式见图9(序列后的注释形式为“n/m”,n表示该编辑形式数量,m表示所挑取的单克隆总数)。挑取6只羔羊共计144个单克隆测序,编辑类型结果(见表2)表明:GM108号羔羊所测19个单克隆均为野生型,而其余羔羊的125个单克隆发生ASIP基因突变的位置均毗邻PAM序列,包含10种突变形式,其突变类型主要以4bp和2bp碱基删除为主,其次为27bp碱基删除,最长删除片段为27bp。删除4bp和2bp碱基的羔羊毛色多呈现黑色或黑白色,着色较深,而未发生突变的GM108号羔羊毛色为白色。
表2 注射ASIP-Cas9mRNA基因编辑羊不同编辑类型统计
综合PCR测序和TA克隆测序结果,在生产的6只羔羊中有5只羔羊发生ASIP基因删除/插入突变,阳性率高达83.33%(见表3),且目前长势良好。
表3 CRISPR/Cas9靶向删除ASIP基因mRNA显微注射生产基因编辑绵羊结果统计
Claims (8)
1.能够特异的靶向修饰绵羊ASIP基因的sgRNA,为序列表的序列4所示的RNA。
2.编码权利要求1中所述sgRNA的DNA分子。
3.一种特异性敲除绵羊ASIP基因的成套核酸分子,包括权利要求1所述的sgRNA。
4.一种特异性敲除绵羊ASIP基因的成套核酸分子,包括权利要求1所述的sgRNA和Cas9mRNA。
5.一种特异性敲除绵羊ASIP基因的成套核酸分子,包括权利要求2所述的DNA分子。
6.一种特异性敲除绵羊ASIP基因的成套核酸分子,包括权利要求2所述的DNA和编码Cas9mRNA的DNA分子。
7.一种特异性敲除绵羊ASIP基因的方法,包括如下步骤:将权利要求1所述的sgRNA和Cas9mRNA共转染绵羊细胞,从而敲除绵羊ASIP基因。
8.一种获得毛色改变的绵羊的方法,包括如下步骤:将权利要求1所述的sgRNA和Cas9mRNA共转染绵羊细胞,从而敲除绵羊ASIP基因,得到毛色改变的绵羊。
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