Construction and Verification of CRISPR/Cas9 System Expression Vector for Mouse X Chromosome Cutting

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

Abstract

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

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.

Figures/Tables 10

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

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

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

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

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

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

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

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

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

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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