基于CRSIPR/Cas9技术构建凝血因子8基因敲除小鼠模型及表型验证

doi:10.13560/j.cnki.biotech.bull.1985.2021-1564

摘要/Abstract

摘要：

基于CRISPR/Cas9技术构建凝血因子8（F8）基因敲除小鼠模型并对其表型进行初步验证。针对F8基因外显子1非编码区和外显子26下游序列设计sgRNA靶位点，体外转录获得sgRNA，与编码Cas9的mRNA混合后通过受精卵显微注射方法获得F₀代阳性敲除小鼠，通过繁育及基因型鉴定获得F8基因敲除纯合子小鼠（F8^-/-小鼠）；通过逆转录PCR（RT-PCR）检测主要组织中F8基因在mRNA水平的表达情况，通过酶联免疫吸附测定（ELISA）检测血浆中F8基因在蛋白水平的表达情况；通过活化部分凝血活酶时间检测（APTT）和纤维蛋白原含量（FIB）测定实验检测F8基因敲除后小鼠凝血功能情况。PCR及测序结果表明F8基因在小鼠基因组中被成功敲除；RT-PCR 和ELISA检测结果显示，F8^-/-小鼠中F8在mRNA和蛋白水平上表达均显著低于野生型小鼠（WT小鼠）（P<0.000 1，P<0.01）；APTT、FIB和滴血实验检测结果显示，F8^-/-小鼠血浆凝固时间显著高于WT小鼠（P<0.05），F8^-/-小鼠存在严重凝血障碍表型，给予人凝血因子Ⅷ后凝血可恢复到正常水平。利用 CRISPR/Cas9技术成功构建F8基因敲除小鼠模型并初步验证其表型，证实该小鼠F8基因的缺失可以导致凝血功能障碍。

关键词: 凝血因子8（F8）, CRISPR/Cas9, 基因敲除, 血友病A

Abstract:

A coagulation factor 8（F8）gene knockout mouse model was constructed based on CRISPR/Cas9 technique and its phenotype was verified. sgRNA target sites were designed according to exon 1 non-coding region to exon 26 downstream sequences of F8 gene，the sgRNA was obtained from in vitro transcription and mixed with mRNA of encoding Cas9. F0 generation positive knockout mice were obtained by microinjection of fertilized eggs. F8^-/- gene knockout homozygous mice（F8^-/- mice）were obtained by breeding and genotype identification. The expression of F8 gene at mRNA level in main tissues was detected by reverse transcription PCR（RT-PCR），the protein expression of F8 gene in plasma was detected by enzyme-linked immunosorbent assay（ELISA），and the coagulation function of F8 knockout mice was detected by activated partial thromboplastin time（APTT）and fibrinogen content（FIB）. PCR and sequencing results showed that F8 gene was knocked out successfully in mouse genome，and RT-PCR and ELISA results demonstrated that the expression of F8 in F8^-/- mice was significantly lower than that in wild-type mice（WT mice）（P < 0.0001，P<0.01）. APTT，FIB and drop blood test results revealed that the plasma coagulation time of F8^-/- mice was significantly longer than that of WT mice. F8^-/- mice had severe coagulation disorder phenotype，which restored to normal levels after administration of human factor VIII. The F8 gene knockout mice model is successfully constructed by CRISPR/Cas9 technique and its phenotype is preliminarily verified，which confirms that the deletion of F8 gene can lead to coagulation dysfunction.

Key words: coagulation factor Ⅷ（F8）, CRISPR/Cas9, gene knockout, hemophilia A

王海杰, 王成稷, 郭洋, 王云, 陈艳娟, 梁敏, 王珏, 龚慧, 沈如凌. 基于CRSIPR/Cas9技术构建凝血因子8基因敲除小鼠模型及表型验证[J]. 生物技术通报, 2022, 38(10): 273-280.

WANG Hai-jie, WANG Cheng-ji, GUO Yang, WANG Yun, CHEN Yan-juan, LIANG Min, WANG Jue, GONG Hui, SHEN Ru-ling. Construction of Coagulation Factor 8 Gene Knockout Mouse Model Based on CRSIPR/Cas9 Technique and Verification of Phenotype[J]. Biotechnology Bulletin, 2022, 38(10): 273-280.

图/表 12

表1 sgRNA序列信息

Table 1 sgRNA sequence information

sgRNA靶序列 sgRNA target sequence	序列信息 Sequence information（5'-3'）
sgRNA 1	AGGCTTAACCCATTTCCTGC TGG
sgRNA 2	TCTGGATCCACAGATATAGC AGG

表2 寡核苷酸链序列信息

Table 2 Oligonucleotide chain sequence information

寡核苷酸OligoDNA	序列信息Sequence information（5'-3'）
sgRNA 1 正义链	ACTTATTAGTTTGCAAAACG
sgRNA 1 反义链	GTGGCCTTCGATTAGGCGGA
sgRNA 2 正义链	CGGTCAGAGCTGCACATACA
sgRNA 2 反义链	GCCAAATTGTGATCTCAGTT

表3 引物序列信息

Table 3 Primer sequence information

引物Primer	序列信息Sequence information（5'-3'）
P1	TTTTGGCTTTCTAACAGGTATCG
P2	CCAGAAAAGGGCATCAGGT

表4 逆转录PCR引物序列

Table 4 Reverse transcription PCR primer sequences

基因 Gene	上游引物信息Upstream primer information（5'-3'）	下游引物信息Downstream primer information（5'-3'）
F8	AGGCCTCATTGGAGCTCTGCTA	CCATTGGGATTCCTCAGGGCA
β-actin	CGTTGACATCCGTAAAGACC	AACAGTCCGCCTAGAAGCAC

图1 F8 -/-小鼠基因敲除策略 Cas9/sgRNA复合体通过识别外显子1-26（exon1-26）区域靶点将F8基因相应片段切除

Fig.1 Knockout strategy of F8 -/- mice The Cas9/sgRNA complex removed the corresponding fragments of the F8 gene by recognizing targets in the exon1-26 region

图2 F8-/-基因敲除小鼠基因型鉴定结果（a）F8-/-基因敲除小鼠的鉴定策略，通过两对引物P1/P2和P3/P4进行PCR鉴定，确定小鼠基因型，引物位置图；（b）和（c）为基因型鉴定电泳图，（b）中利用P1/P2引物对进行PCR检测，WT小鼠不能获得条带，F8-/+杂合子小鼠与F8-/-纯合子小鼠获得单一772bp片段，（c）中P3/P4引物对进行PCR检测，WT小鼠与F8-/+杂合子小鼠可获得357bp片段，F8-/-纯合子小鼠不能获得条带。经电泳鉴定，（b）和（c）中WT小鼠编号为3，5，6，11，F8-/+杂合子小鼠编号为1，2，7，8，9，10，14，16，18，19，F8-/-纯合子小鼠编号为4，12，13，15，17，20。WT：野生型；M：1kb DNA maker

Fig.2 Genotype results of F8-/- gene knockout mice （a）The identification strategy of F8-/- knockout mice. Two pairs of primers P1/P2 and P3/P4 were used for PCR identification to determine the mice genotype. The primer positions were shown in the figure.（b）and（c）were genotype identification electrophoresis diagram，with P1/P2 primer pair for PCR detection in（b），bands were obtained in WT mice，F8-/+ heterozygous mice and F8-/- homozygous mice obtained a single 772 bp fragment.（c）The P3/P4 primer pair was used for the PCR detection，WT mice and F8-/+ heterozygous mice could obtain a 357 bp fragment，F8-/- homozygous mice could not obtain a band. According to electrophoresis identification，WT mice in（b）and（c）were numbered 3，5，6，11，and F8-/+ heterozygous mice were numbered 1，2，7，8，9，10，14，16，18，19，F8-/- homozygous mice were numbered 4，12，13，15，17，20. WT：Wild type mice. M：1 kb DNA maker

表5 WT小鼠和F8-/-敲除小鼠的序列信息

Table 5 Sequence information of wild type and F8-/-mice genotype

类型Index	序列信息Sequence information（5'-3'）
野生型小鼠 WT mice	AGGATTCAAACTTGTTAGGATGCACCCAGCAGGA- AATGGGTTAAGCCTTAGCTCAGCCACTCTTCCTA- TTCCAGTT……GAGAAGTTGCTGAGAGTTCTATA- TCTGGATCCACAGATATAGCAGGAAGAGAAAGA- CACTGGGACTGACTTGGG
F8^-/-小鼠序列 F8^-/-mice	AGGATTCAAACTTGTTAGGATGCACCCAGCAGGAA …（207293 bp缺失）…GAGAAAGACACTGGGAC- TGACTTGGG

图3 F8 -/-小鼠F8 mRNA 相对表达水平（a）F8基因在WT小鼠各个主要组织中的表达情况；（b）F8 -/-和WT小鼠心、肝、脾、肺和肾组织中F8 mRNA相对表达水平检测，每组3 只小鼠（****P <0.0001）；（c）RT-PCR代表性电泳鉴定结果（肝组织）；WT-野生型，F8 -/+-杂合子，F8 -/--纯合子

Fig.3 Analysis of relative mRNA expression in F8-/- mice （a）F8 gene expression in multiple tissues of WT mice.（b）F8 mRNA relative expression level detection in heart，liver，spleen，lung and kidney tissues of F8 -/- and WT mice，3 mice per group（****P <0.0001）.（c）RT-PCR representative identification result of electrophoresis（liver tissue）. WT：Wild type mice，F8 -/+-heterozygous，F8 -/--homozygous mice

图4 F8-/-小鼠F8蛋白相对表达水平 F8 -/-小鼠和WT小鼠血浆中F8蛋白的含量结果，每组5 只小鼠（**P <0.01），WT-野生型，F8 -/--纯合子

Fig.4 Relative expression of F8 protein in F8-/- mice F8 -/- protein content in plasma of F8-/- and WT mice，5 mice per group（**P <0.01），WT-wild type，F8-/-- homozygous

图5 F8 -/-小鼠的APTT A：小鼠凝血功能检测给药过程示意图；B：F8 -/-小鼠与WT小鼠血浆中FIB含量比较；C：F8 -/-小鼠与WT小鼠给予人凝血因子Ⅷ前后APTT值比较；每组5 只小鼠（*P <0.05，****P <0.0001），NS-尾静脉注射生理盐水，APTT-尾静脉注射人凝血因子Ⅷ，WT-野生型，F8 -/--纯合子

Fig.5 APTT results of F8-/- mice A：Schematic diagram of the administration process of blood coagulation in mice. B：Comparison of FIB in plasma of F8 -/- and WT mice. C：Comparison of APTT values before and after administration of human coagulation factor Ⅷ between F8 -/- and WT mice，5 mice per group（*P <0.05，****P <0.0001），to NS-tail vein injected with saline，to APTT-tail vein injected with human coagulation factor Ⅷ，WT-wild type，and F8 -/-- homozygous

图6 F8-/-小鼠滴血实验 A：F8 -/-小鼠注射生理盐水；B：F8 -/-小鼠注射凝血因子

Fig. 6 F8 -/- mice blood drop test A：F8 -/-mice injection of saline. B：F8 -/-mice injection of clotting factor

图7 F8 -/-小鼠的血斑灰度值

Fig.7 Gray value of blood spot of F8-/- mice

参考文献 20

[1]	Jankowska KI, Chattopadhyay M, Sauna ZE, et al. A foundational study for normal F8-containing mouse models for the miRNA regulation of hemophilia A:identification and analysis of mouse miRNAs that downregulate the murine F8 gene[J]. Int J Mol Sci, 2020, 21(16):5621. doi: 10.3390/ijms21165621 URL
[2]	王典文, 涂传清. 血友病A发病分子机制的研究现状[J]. 医学综述, 2013, 19(24):4430-4433.
	Wang DW, Tu CQ. Research status of molecular pathogenesis of hemophilia A[J]. Med Recapitul, 2013, 19(24):4430-4433.
[3]	许正平, 王恩多. 凝血因子Ⅷ[J]. 生物化学与生物物理进展, 1991(4):8-11.
	Xu ZP, Wang ED. Coagulation factor Ⅷ[J]. Prog Biochem Biophys, 1991(4):8-11.
[4]	侯玉香, 肖小璞. 血友病基因治疗概述[J]. 中国输血杂志, 2007, 20(1):71-74.
	Hou YX, Xiao XP. Summary of gene therapy for hemophilia[J]. Chin J Blood Transfus, 2007, 20(1):71-74.
[5]	Yamaguti-Hayakawa GG, Ozelo MC. Gene therapy:paving new roads in the treatment of hemophilia[J]. Semin Thromb Hemost, 2019, 45(7):743-750. doi: 10.1055/s-0039-1688445 pmid: 31096314
[6]	Rangarajan S, Walsh L, Lester W, et al. AAV5-factor VIII gene transfer in severe hemophilia A[J]. N Engl J Med, 2017, 377(26):2519-2530. doi: 10.1056/NEJMoa1708483 URL
[7]	Batty P, Lillicrap D. Advances and challenges for hemophilia gene therapy[J]. Hum Mol Genet, 2019, 28(R1):R95-R101. doi: 10.1093/hmg/ddz157
[8]	Monahan PE, Samulski RJ, Tazelaar J, et al. Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia[J]. Gene Ther, 1998, 5(1):40-49. pmid: 9536263
[9]	Peyvandi F, Garagiola I. Clinical advances in gene therapy updates on clinical trials of gene therapy in haemophilia[J]. Haemophilia, 2019, 25(5):738-746. doi: 10.1111/hae.13816 pmid: 31282050
[10]	Chen HN, Shi M, Gilam A, et al. Hemophilia a ameliorated in mice by CRISPR-based in vivo genome editing of human factor VIII[J]. Sci Rep, 2019, 9(1):16838. doi: 10.1038/s41598-019-53198-y URL
[11]	Guo T, Feng YL, Xiao JJ, et al. Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing[J]. Genome Biol, 2018, 19(1):170. doi: 10.1186/s13059-018-1518-x pmid: 30340517
[12]	Hooper ML. Embryonic stem cells:introducing planned changes into the animal germline[M]. Chur: Harwood Academic Publishers, 1992.
[13]	Lin HF, Maeda N, Smithies O, et al. A coagulation factor IX-deficient mouse model for human hemophilia B[J]. Blood, 1997, 90(10):3962-3966. pmid: 9354664
[14]	陈永昌, 牛昱宇, 季维智. 通过CRISPR/Cas9和TALENs介导的基因打靶技术获得基因修饰的猴模型[J]. 中国细胞生物学学报, 2014, 36(5):557-560.
	Chen YC, Niu YY, Ji WZ. The genetically modified monkey model was obtained by gene targeting mediated by CRISPR/ Cas9 and TALENs[J]. Chin J Cell Biol, 2014, 36(5):557-560.
[15]	Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121):819-823. doi: 10.1126/science.1231143 pmid: 23287718
[16]	梁振伟, 饶书权, 沈岩, 等. 通过CRISPR/Cas9系统敲除人源PDE10A基因[J]. 基础医学与临床, 2014, 34(4):439-443.
	Liang ZW, Rao SQ, Shen Y, et al. Knocking out human PDE10A gene by CRISPR/Cas9 system[J]. Basic Clin Med, 2014, 34(4):439-443.
[17]	郑武, 谷峰. CRISPR/Cas9的应用及脱靶效应研究进展[J]. 遗传, 2015, 37(10):1003-1010.
	Zheng W, Gu F. Progress of application and off-target effects of CRISPR/Cas9[J]. Hereditas, 2015, 37(10):1003-1010.
[18]	Ma YW, Chen W, Zhang X, et al. Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing in rats by inhibiting NHEJ and using Cas9 protein[J]. RNA Biol, 2016, 13(7):605-612. doi: 10.1080/15476286.2016.1185591 pmid: 27163284
[19]	Du J, Yin NR, Xie T, et al. Quantitative assessment of HR and NHEJ activities via CRISPR/Cas9-induced oligodeoxynucleotide-mediated DSB repair[J]. DNA Repair(Amst), 2018, 70:67-71.
[20]	汪启翰, 怀聪, 孙瑞林, 等. 利用CRISPR/Cas系统快速高效构建血友病乙小鼠模型[J]. 遗传, 2015, 37(11):1143-1148.
	Wang QH, Huai C, Sun RL, et al. A quick and efficient method to generate hemophilia B mouse models by the CRISPR/Cas system[J]. Hereditas, 2015, 37(11):1143-1148.