切割小鼠X染色体的CRISPR/Cas9系统表达载体的构建及验证

doi:10.13560/j.cnki.biotech.bull.1985.2020-0963

摘要/Abstract

摘要：

性别控制技术在畜牧业生产、濒危动物保护、伴性疾病预防等方面都有着重要的意义,但是目前较为有效的精子分离技术却因成本高昂、技术难度大的问题无法广泛应用于畜禽生产上。受在蚊子上成功实现性别控制的研究启发,本实验设计了两个靶向小鼠X染色体重复序列的sgRNA,构建了含有靶向切割小鼠X染色体的CRISPR/Cas9系统的CMV载体,并对其进行体外、细胞及胚胎水平上的切割效率验证。结果显示：sgRNA X1和sgRNA X3体外介导的Cas9蛋白切割靶序列的效率分别为60.0%和75.0%;CMV载体成功转染MLTC-1细胞后,死亡率与对照组（转染空载体）相比显著提高;CMV载体对X1靶位点的突变效率为11.1%;与对照组相比,小鼠孤雌胚胎注射CMV载体后囊胚率有一定的下降,但未达到显著水平,而囊胚细胞数则显著下降（P<0.01）。实验证明了CMV载体在体外、细胞及胚胎水平上均具有较高的切割效率,能够成功删除X染色体,可用于减少某一性别的精子或胚胎,从而实现哺乳动物的性别控制。

关键词: 性别控制, CRISPR/Cas9, X染色体, 小鼠

Abstract:

Gender control technology plays an important role in animal husbandry production,endangered animal protection,and prevention of sexually transmitted diseases. However,currently effective sperm separation technology cannot be widely used in animal production due to its high cost and technical difficulty. Inspired by the successful implementation of gender control in mosquitoes,this experiment designed two sgRNAs targeted at the X chromosome repeat sequence of mice,constructed a CMV vector containing the CRISPR/Cas9 system to target the X chromosome of mice,and verified its cutting efficiency at the level of in vitro,cell and embryo. The results showed that the efficiency of Cas9 protein mediated by sgRNA X1 and sgRNA X3 in vitro was 60.0% and 75.0% respectively. After CMV vector was successfully transfected into MLTC-1 cells,the mortality rate was significantly higher than that of the control group（transfected with empty vector）. The mutation efficiency of CMV vector to X1 target was 11.1%. Compared with the control group,the blastocyst rate of parthenogenetic embryos of mice to some extent after CMV injection decreased,but not to a significant level,while the number of blastocysts decreased significantly（P < 0.01）.This experiment has proved that CMV vector demonstrates a high cutting efficiency in vitro,cell and embryo levels,and can successfully delete X chromosomes,which can be used to reduce sperm or embryos of a certain sex,thus achieving mammalian sex control.

Key words: gender control, CRISPR/Cas9, X chromosome, mice

谢绍怡, 蒋璐蔓, 杨晓峰, 张仙玉, 吴珍芳, 李紫聪. 切割小鼠X染色体的CRISPR/Cas9系统表达载体的构建及验证[J]. 生物技术通报, 2021, 37(5): 67-75.

XIE Shao-yi, JIANG Lu-man, YANG Xiao-feng, ZHANG Xian-yu, WU Zhen-fang, LI Zi-cong. Construction and Verification of CRISPR/Cas9 System Expression Vector for Mouse X Chromosome Cutting[J]. Biotechnology Bulletin, 2021, 37(5): 67-75.

图/表 10

表1 靶片段扩增引物

Table 1 Target fragment amplification primers

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	产物长度 Product size/bp
X1-F	TGAGTACTTGGTTAGGGTGAGC	1 212
X1-R	TGGAAGTCTCTGGCATA	1 212
X3-F	AAGGTTCCCAATGGTCACAGG	1 383
X3-R	ACCATACCATGGTTTTCCCCA	1 383

表2 sgRNA体外转录模板扩增引物

Table 2 Primers for amplification of sgRNA transcription template in vitro

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	产物长度 Product size/bp
sgRNA X1-F	TAATACGACTCACTATAGGGG- CTTGGTTAGGGTGAGTGCT	120
sgRNA X1-R	AAAAGCACCGACTCGGTGCC	120
sgRNA X3-F	TAATACGACTCACTATAGGG- TAAGTGCTGTGTGCTGCTAC	120
sgRNA X3-R	AAAAGCACCGACTCGGTGCC	120

表3 X1靶位点扩增引物

Table 3 Primers for amplification of the X1 target site

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	产物片段 Product size/bp
X1 mutation-F	GCATGCTTGGTTAGGGTGAGTT	954
X1 mutation-R	GCTGGAAACCCAAGCAC	954

表4 靶向小鼠X染色体多拷贝基因的sgRNA序列

Table 4 Sequences of sgRNA targeting mouse X chromosome multi-copy genes

sgRNA名称 sgRNA name	靶位点（拷贝数） Target location（copy number）	核苷酸序列 Nucleotide sequence（5'-3'）
sgRNA X1	X1（72）	GCTTGGTTAGGGTGAGTGCT
sgRNA X3	X3（99）	TAAGTGCTGTGTGCTGCTAC

图1 CMV载体的构建及鉴定 A：CMV载体构建示意图。B：载体一和sgRNA片段合成质粒双酶切结果电泳图,M1为1000 DNA marker,M2为10000 DNA Marker;a为原载体一,b为酶切后的载体一;M为10000 DNA marker;pKMV为sgRNA片段合成质粒

Fig.1 Construction and identification of CMV vector A: Schematic diagram of CMV carrier construction. B: Electrophoresis diagram of plasmid double digestion results synthesized by vector 1 and sgRNA fragments, M1 is 1000 DNA marker, M2 is 10000 DNA marker, A is the original vector 1, B is the digested vector 1, M is 10000 DNA marker, and pKMV is a plasmid synthesizing sgRNA fragment

图2 sgRNA X1和sgRNA X3介导Cas9蛋白在体外切割靶序列效率验证的电泳图图中a为未切割的sgRNA靶序列片段,b为切割后的sgRNA靶序列片段,箭头指示为切割后片段,M为2000 DNA marker

Fig. 2 Electrophoresis diagram of verifying the efficiency of sgRNA X1 and sgRNA X3 mediated Cas9 protein cleaving target sequence in vitro a is the uncut sgRNA target sequence fragment, b is the cut sgRNA target sequence fragment, arrow indicates the cut fragment, and M is 2000 DNA marker

图3 CMV载体、空载体转染MLTC-1细胞不同时间的荧光细胞数比较结果 A：雄性细胞转染CMV载体和空载体后不同时间点的荧光效果图;B：转染载体后不同时间点绿色荧光细胞的比例

Fig.3 Comparison results of the number of fluorescent cells in MLTC-1 cells at different time after transfecting with CMV vector and empty vector A: Fluorescence renderings at different time points after male cells were transfected with CMV vector and empty vector. B: Proportion of green fluorescent cells at different time points after vector transfection

图4 CMV载体转染后对MLTC-1细胞的单克隆细胞团X1靶位点处测序结果红色框标注的为靶位点,*为匹配碱基,缺失空白处为突变碱基

Fig. 4 Sequencing results of the X1 target site of the monoclonal cell mass of MLTC-1 cells after CMV vector transfection Target sites are marked in the red box, * is the matching base, and the missing space is the mutation base

表5 CMV载体转染后MLTC-1细胞X1靶位点的突变效率

Table 5 Mutation efficiency of CMV vector transfected MLTC-1 cell X1 target site

转染细胞 Transfection cell	检测筛选获得的单克隆细胞团数 Number of monoclonal cell clusters obtained by screening was detected	突变的单克隆细胞团数 Number of mutant monoclonal cell clusters	突变效率 Mutation efficiency/%
MLTC-1	36	4	11.1

表6 CMV载体和空载体注射孤雌胚体外发育效率比较

Table 6 Comparison of in vitro development efficiency of parthenogenetic embryos injected with CMV vector andempty vector

组别 Group	注射胚胎数Number of injected embryos	卵裂胚数Number of cleavage embryo	卵裂率 Cleavage rate	囊胚数Number of blastocysts	囊胚率 Blastocyst rate	囊胚细胞数 Number of blastocyst cells
注射 CMV 载体组 The Group of CMV vector injection	98	96	98.0%	57	58.1% ^a	33.93±4.93^a
注射空载体组 The Group of empty vector injection	92	88	95.7%	64	69.5% ^a	44.66±6.18 ^b

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