WO2020056661A1 - 快速基因编辑构建灵长类动物疾病模型的方法 - Google Patents

快速基因编辑构建灵长类动物疾病模型的方法 Download PDF

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WO2020056661A1
WO2020056661A1 PCT/CN2018/106658 CN2018106658W WO2020056661A1 WO 2020056661 A1 WO2020056661 A1 WO 2020056661A1 CN 2018106658 W CN2018106658 W CN 2018106658W WO 2020056661 A1 WO2020056661 A1 WO 2020056661A1
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constructing
gene editing
plasmid
primate
disease model
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赵永祥
阳诺
周素芳
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赵永祥
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Definitions

  • the present invention relates to the field of animal models of human diseases, and more particularly, to a method for constructing a primate disease model by rapid gene editing.
  • mice and rats have become the most commonly used model animals in biomedicine.
  • rodents and humans have relatively long kinships and large species differences, which limits their effectiveness in simulating the development of human diseases.
  • non-human primates such as cynomolgus monkeys
  • primates are highly similar to humans in terms of genetic evolution, nerves, physiology, immunity, and gene sequence, so primates are the study of human genes. The most valuable model animal for function and disease.
  • mouse tumor models are mainly based on the gene targeting technology of embryonic stem cells or the technology of somatic cell nuclear transfer, which is achieved by knocking out or knocking in the target genes, and precisely modifying the specific genes of transgenic animals. .
  • the CRISPR-CAS9 system has attracted much attention as a new gene editing tool. It is mainly based on an adaptive immune system modified by bacteria. When foreign DNA invades, CRISPR-RNA guides the specific cleavage of CAS protein, resulting in double strand breaks (DSBs) at the DNA® site. ), After DNA damage
  • DSBs activate two different repair mechanisms, non-homologous ending-joining (NHEJ) or homologous recombination (HR), which are inherent in cells, to repair damaged DNA, thereby achieving site-specific targeting of the genome. edit.
  • NHEJ non-homologous ending-joining
  • HR homologous recombination
  • the technical problem to be solved by the present invention is to provide a method for constructing a primate disease model quickly by gene editing in response to the problem that the primate has a long period of tumor model establishment and high cost.
  • the technical solution of the present invention to solve the above technical problem is to provide a method for rapid gene editing to construct a primate disease model for non-diagnostic or therapeutic purposes.
  • the method includes the following steps:
  • step (b) using a biopsy puncture needle to inject the sgRNA expression plasmid prepared in step (a) into the portal vein of the liver of a primate until liver cells become cancerous, and obtain a model.
  • step (a) further includes:
  • the step (a4) comprises: taking a logarithmic growth phase of COS-7 cells and laying a six-well plate with 1.5 x cells per well 106, when the cell density reached 70%, using the transfection reagent Lipofectamine 3000 transfection, each well was transfected with a plasmid expressing sgRNA The amount was controlled at 2.5-3. (Vg, cells were harvested 48 hours after transfection, and genomic DNA was extracted according to the procedure of the genome extraction kit.
  • the gRNA oligonucleotide includes a base sequence 5 '-CAATTCTGCCCTCACAGCTC-3'.
  • the step (b) includes:
  • the injection volume is controlled within 400 pL;
  • the method for constructing a primate disease model by rapid gene editing after step (b5), the method further comprises: 45 days later, the target site is amplified and the amplified fragment is purified, and the target site is Perform deep sequencing to analyze gene editing.
  • the method for constructing a primate disease model by the rapid gene editing and the sgRNA for specifically targeting the P53 tumor suppressor gene in primate liver cells have the following beneficial effects:
  • the sgRNA expression plasmid constructed by the pX330 plasmid can be directly injected into the liver tissue of primates, thereby quickly constructing a tumor model.
  • FIG. 1 is a schematic diagram of pX330 plasmid structure and gRNA insertion position
  • FIG. 2 is a schematic diagram of gene editing in a deep sequencing analysis of a target site 45 days after liver puncture in a cynomolgus monkey Invention Examples
  • the method for constructing a primate disease model by rapid gene editing according to the present invention includes the following steps:
  • the gRNA oligonucleotide is selected at the position of the first exon near the ATG, and is designed according to the design principle of N20NGG.
  • the gRNA oligonucleotide is single-stranded and includes the base sequence 5 ’
  • gRNA oligonucleotides were synthesized by GENEWIZ Inc. of Suzhou.
  • the pX330 plasmid expressing Cas9 protein and sgRNA was purchased from Addgene (Addgene, Cambridge, MA, USA).
  • the pX330 plasmid is first cut with a Bbs I restriction enzyme, and then recovered by PCR cleaning and recovery kit (purchased from Axygen Inc. (Axygen Inc., USA)); Acid single-strand annealing to form a double-strand, linked to Bbs
  • the same method can also be used to construct a control plasmid pX330-GFP-sgR NA for the GFP gene.
  • an in vitro cell test may be performed first, which specifically includes the following steps:
  • DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin, 100 pg / mL streptomycin), 3TC, 5% C02 cell culture incubator. Within routine culture. DMEM medium, fetal calf serum and penicillin were purchased from Gibco (GIBCO, Thermo Fisher Scientific Inc., Waltham, MA, USA).
  • COS-7 cells in logarithmic growth phase were taken and plated in a six-well plate, and cells in each well of the six-well plate were 1.5 x 10 6 .
  • the amount of sgRNA expression plasmid pX330-p53-sgRNA transfected in each well was controlled to 2.5-3.0, and the equivalent amount of pX330-gRNA-GFP vector was used as a negative control .
  • Cells were harvested 48 hours after transfection, and genomic DNA was extracted according to the steps of the genome extraction kit.
  • the COS-7 genomic DNA is used as a template for amplification target sites, and p53-F and p53-R are used as primers.
  • the reaction conditions are as follows
  • the target site sequence was amplified with Q5 enzyme, ligated to the T vector, and the ligated product was transformed into competent cells, and 30 monoclonal clones were randomly selected. If the p53 gene in COS cells The presence of a base insertion or deletion mutation at the target site indicates a genetic mutation.
  • a healthy male cynomolgus monkey 5 to 8 years old, weighing 3.2-6.0kg, can be selected and raised at the Guangxi Fangchenggang Changchun Biotechnology Development Co., Ltd. cynomolgus monkey medical application research base.
  • This research The base has passed AAALAC (Assessment and Accreditation Of Laboratory Animal Care) certification.
  • AAALAC Accreditation and Accreditation Of Laboratory Animal Care
  • injection volume was controlled within 400 ⁇ iL.
  • 1 ml of 0.9% sodium chloride injection was rapidly injected.
  • Ultrasound showed a strong echo-diffused sound image, which confirmed that the portal vein injection of the CRISPR-Cas plasmid system was successful.
  • the alcohol injection therapy needle is withdrawn from the body under real-time ultrasound observation. After all operations were completed, the liver and perihepatic were scanned by ultrasound to exclude bleeding and organ damage.
  • the target site was amplified and the amplified fragment was purified, and the target site was subjected to deep sequencing to analyze gene editing.
  • deep sequencing 6 cynomolgus monkeys in the experimental group detected mutations near the PAM region of the gRNA target site, with a mutation rate of 50%.
  • the mutations included insertion of nucleic acid sequences and deletion of nucleic acids.
  • Software analysis showed that the length of the inserted or deleted nucleotides ranged from lbp to 20bp, with 1 base deletion and insertion, and 20 base deletion and insertion.
  • the results of deep sequencing were analyzed.
  • the frequency of Indel of the p53 gene target site in the liver tissue of the six cynomolgus monkeys was 5.39%.
  • A is one of the representatives of healthy cynomolgus monkeys;
  • B is one of the representatives of cynomolgus monkeys injected with the control plasmid pX330-GFP-sgRNA;
  • C is injection of sgRNA expression plasmid pX330-p53-sgRNA cynomolgus One of the monkeys.
  • C. cynomolgus monkeys showed p53-positive cells, CK19-positive cells, and Ki67-positive cells, as shown by the circles in the figure.
  • liver cancer cells C cynomolgus monkeys, biliary epithelial cells, P 53, Ki67 positive rate was significantly higher than A and B cynomolgus cynomolgus monkeys.
  • Bile duct epithelial cells were strongly positive for CK19, significantly higher than cynomolgus monkeys and B. cynomolgus monkeys.

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Abstract

提供了一种快速基因编辑构建灵长类动物疾病模型的方法，所述方法包括以下步骤：（a）使用gRNA寡核苷酸及pX330质粒构建sgRNA表达质粒；（b）使用活检穿刺针将所述步骤（a）中制备的sgRNA表达质粒注射到灵长类动物肝脏门静脉内，直至肝脏细胞出现癌变，获取模型。通过由gRNA寡核苷酸及pX330质粒构建的sgRNA表达质粒，可直接注射到灵长类动物的肝脏组织，从而快速构建肿瘤模型。

Description

快速基因编辑构建灵长类动物疾病模型的方法 技术领域
[0001] 本发明涉及人类疾病动物模型领域， 更具体地说， 涉及一种快速基因编辑构建 灵长类动物疾病模型的方法。
背景技术
[0002] 建立动物疾病模型是研究人类肿瘤的发病机制、 药物筛选与疫苗开发的必要工 具。 由于啮齿类动物具有个体小、 繁殖迅速、 遗传背景清楚、 转基因技术成熟 等优势， 小鼠、 大鼠等啮齿类动物就成为生物医学最常用的模式动物。 但啮齿 类动物和人的亲缘关系比较远、 种属差异大， 这就限制了其在模拟人类疾病发 生发展时的有效性。 相对小鼠、 大鼠而言， 非人灵长类动物， 如食蟹猴等， 在 遗传进化、 神经、 生理、 免疫和基因序列等方面与人类高度近似， 所以灵长类 动物是研究人类基因功能及疾病最有价值的模式动物。
[0003] 目前， 小鼠肿瘤模型的构建主要是基于胚胎干细胞的基因打靶技术或是体细胞 核移植技术， 其通过对目标基因的敲除或敲入、 对特定基因进行精确修饰的转 基因动物而实现。
[0004] 然而， 在灵长类动物中， 由于胚胎干细胞和体细胞核移植技术尚不成熟， 因此 ， 很难采用同样的技术策略获得精确基因修饰的动物疾病模型。 传统的方法是 通过对生殖细胞进行基因修饰来构建动物模型， 但是灵长类动物食蟹猴的性成 熟时间与繁殖周期长， 花费代价高。
[0005] CRISPR-CAS9系统作为一种新的基因编辑工具备受关注。 它主要是基于细菌的 一种获得性免疫系统改造而成， 当外源 DNA入侵时， CRISPR-RNA指导 CAS蛋 白特异性剪切， 在 DNA®位点产生 DNA双链断裂 (Double strand breaks， DSBs ) ， DNA损伤后产生的
DSBs激活细胞内固有的非同源末端连接 (Non-homologous ending-joining, NHEJ) 或同源重组 (Homologous recombination , HR)两种不同的修复机制对损伤 的 DNA进行修复， 从而实现对基因组的定点编辑。 [0006] 然而， 在与人类遗传背景最接近的灵长类大动物—食蟹猴体内的体细胞水平 上， 尚无法使用 CRISPR技术来建立诱导疾病模型。
技术问题
[0007] 本发明要解决的技术问题在于， 针对上述灵长类动物建立肿瘤模型周期长、 花 费代价高的问题， 提供一种快速基因编辑构建灵长类动物疾病模型的方法。 问题的解决方案
技术解决方案
[0008] 本发明解决上述技术问题的技术方案是， 提供一种快速基因编辑构建灵长类动 物疾病模型的方法， 用于非诊断或治疗目的， 所述方法包括以下步骤：
[0009] (a) 使用 gRNA寡核苷酸及 pX330质粒构建 sgRNA表达质粒；
[0010] (b) 使用活检穿刺针将所述步骤 (a) 中制备的 sgRNA表达质粒注射到灵长类 动物肝脏门静脉内， 直至肝脏细胞出现癌变， 获取模型。
[0011] 本发明所述的快速基因编辑构建灵长类动物疾病模型的方法中， 所述步骤 (a
) 包括：
[0012] (al) 将 pX330质粒用 Bbs I限制性内切酶切割后,用 PCR清洁回收试剂盒回收； [0013] (a2) 使 gRNA寡核苷酸单链退火形成双链， 连接至 Bbs機性化的 pX330质粒
， 获得 sgRNA表达质粒。
[0014] 本发明所述的快速基因编辑构建灵长类动物疾病模型的方法中， 所述步骤 (a ) 还包括：
[0015] (a3) 对 COS-7细胞使用 DMEM培养基进行细胞培养；
[0016] (a4) 取对数生长期的 COS-7细胞， 使用 Lipofectamine 3000转染试剂转染， 并 在转染完成按照基因组提取试剂盒操作步骤提取基因组 DNA;
[0017] (a5) 使用 Q5酶对提取的基因组 DNA扩增靶位点， 并进行 T7E1酶切检测及 TA 克隆测序， 在确认基因组 DNA中 P53基因靶位点序列产生基因突变后执行步骤 ( b) °
[0018] 本发明所述的快速基因编辑构建灵长类动物疾病模型的方法中， 所述步骤 (a4 ) 包括： 取对数生长期的 COS-7细胞铺六孔板， 每孔细胞 1.5 x 10 6, 当细胞密度 达到 70%时,使用 Lipofectamine 3000转染试剂转染， 每个孔转染 sgRNA表达质粒 的量控制在 2.5-3.(Vg， 转染后 48小时收细胞， 按照基因组提取试剂盒操作步骤提 取基因组 DNA。
[0019] 本发明所述的快速基因编辑构建灵长类动物疾病模型的方法中， 所述 gRNA寡 核苷酸包括碱基序列 5’ - CAATTCTGCCCTCACAGCTC - 3’。
[0020] 本发明所述的快速基因编辑构建灵长类动物疾病模型的方法中， 所述步骤 (b ) 包括：
[0021] (bl) 在 B超引导下， 定位门静脉左支矢状部， 选择安全进针路径， 经腹部皮 肤进针后， 实时观察针尖位置， 将酒精注射疗法针引导到达门静脉左支矢状部 前壁前方， 当有突破感且回抽见血后即可确定针尖成功进入门静脉管腔；
[0022] (b2) 向管腔内快速推注 120 ug pX330-p53-sgRNA或对照质粒
pX330-EGFP-sgRNA , 注射体积控制在 400 pL内；
[0023] (b3) 快速推注 0.9%氯化钠注射液 lml， 超声可见强回声弥散声像， 证实经门 静脉注射 CRISPR-Cas质粒系统成功；
[0024] (b4) 推注完成后， 在超声实时观察下， 将酒精注射疗法针退出体外；
[0025] (b5) 所有操作完成后， 超声扫查肝脏及肝周， 排除出血及脏器损伤。
[0026] 本发明所述的快速基因编辑构建灵长类动物疾病模型的方法中， 所述步骤 (b5 ) 之后还包括： 45天后， 扩增靶位点并纯化扩增片段， 对靶位点进行深度测序 分析基因编辑情况。 发明的有益效果
有益效果
[0027] 本发明的快速基因编辑构建灵长类动物疾病模型的方法及用于特异性靶向灵长 类动物肝脏细胞 P53抑癌基因的 sgRNA具有以下有益效果： 通过由 gRNA寡核苷 酸及 pX330质粒构建的 sgRNA表达质粒， 可直接注射到灵长类动物的肝脏组织， 从而快速构建肿瘤模型。
对附图的简要说明
附图说明
[0028] 图 1是 pX330质粒结构及 gRNA插入位置示意图；
[0029] 图 2是食蟹猴肝脏穿刺 45天后对靶位点进行深度测序分析基因编辑情况示意图 发明实施例
本发明的实施方式
[0030] 为了使本发明的目的、 技术方案及优点更加清楚明白， 以下结合附图及实施例 ， 对本发明进行进一步详细说明。 应当理解， 此处所描述的具体实施例仅仅用 以解释本发明， 并不用于限定本发明。
[0031] 本发明快速基因编辑构建灵长类动物疾病模型的方法， 用于非诊断或治疗目的 ， 包括以下步骤：
[0032] 1.使用 gRNA寡核苷酸及 pX330质粒构建 sgRNA表达质粒
[0033] 上述 gRNA寡核苷酸选择在第一个外显子靠近 ATG的位置,按照 N20NGG的设 计原则设计， 该 gRNA寡核苷酸为单链并包括碱基序列 5’
-CAATTCTGCCCTCACAGCTC - 3， (其反向序列 5’ -GAGCTGTGAGGGCAG AATTG - 3’ ) 。 上述 gRNA寡核苷酸由苏州金唯智公司 (GENEWIZ Inc.) 合成 。 表达 Cas9蛋白和 sgRNA的 pX330质粒购买于 Addgene公司 (Addgene, Cambridge, MA , USA)。
[0034] 在该步骤中， 首先将 pX330质粒用 Bbs I限制性内切酶切割后,用 PCR清洁回收试 剂盒 (购自 Axygen公司 (Axygen Inc., USA)) 回收； 然后使 gRNA寡核昔酸单链退 火形成双链， 连接至 Bbs
I线性化的 pX330质粒， 获得 sgRNA表达质粒 pX330-p53-sgRNA。 如图 1所示， U6 启动 gRNA转录， CBh启动子启动 Cas9蛋白表达， NLS是核定位信号。
[0035] 该步骤中还可采用同样的方法， 针对 GFP基因， 构建对照质粒 pX330-GFP-sgR NA。
[0036] 为验证上述 sgRNA表达质粒 pX330-p53-sgRNA是否构件成功， 可先进行体外细 胞检测， 具体包括以下步骤：
[0037] ( 1) 对 COS-7细胞使用 DMEM培养基进行细胞培养。 上述 cos-7细胞购自上海 中科院细胞库， 细胞培养使用 DMEM培养基 (含有 10 %胎牛血清和 100 U/mL青 霉素、 lOO pg/mL链霉素) ， 3TC、 5% C02的细胞培养箱内常规培养。 DMEM 培养基、 胎牛血清和青链霉素购自美国 Gibco公司 (GIBCO, Thermo Fisher Scientific Inc., Waltham, M A, USA)。
[0038] (2) 取对数生长期的 COS-7细胞使用 Lipofectamine 3000转染试剂转染， 并在 转染完成按照基因组提取试剂盒操作步骤提取基因组 DNA。 Lipofectamine 3000 车专染试齐1 J购自美国 Invitrogen公司 (Invitrogen, Thermo Fisher Scientific Inc.,
Waltham, MA, USA)。
[0039] 在进行转染时， 取对数生长期的 COS-7细胞铺六孔板， 六孔板的每孔细胞 1.5 x 10 6。 当细胞密度达到 70%时,使用 Lipofectamine 3000转染试剂转染， 每个孔转 染 sgRNA表达质粒 pX330-p53- sgRNA的量控制在 2.5-3.0 ， 以等量 pX330-gRNA-GFP载体为阴性对照。 转染后 48小时收细胞， 按照基因组提取试剂盒操作步骤提取基因组 DNA。
[0040] (3) 使用 Q5酶对提取的基因组 DNA扩增靶位点， 并进行 T7E1酶切检测及 TA 克隆测序， 在确认基因组 DNA中 P53基因靶位点序列产生基因突变后执行后续步 骤。
[0041] 扩增靶位点以 COS-7基因组 DNA为模板， p53-F和 p53-R为引物， 反应条件如下
: 98°C, 30s； 35个循环 (98°C， 10s； 60°C, 15s； 72°C, 20s) , 72°C, 2min； 95 °C, 5min； -2°C/s降温至 85°C; -0.1°C/s从 85°C降温至 25°C。 在扩增靶位点后， 立刻使用 PCR清洁回收试剂盒回收， 并将回收产物 1(VL， 加入 0.5pL T7El酶 (购 自 NEB公司 (New England Biolabs, USA)) ， 37°C酶切 30 min。 用 2%琼脂糖凝胶 电泳分析。 用 Q5酶扩增靶位点序列， 连接至 T载体， 连接产物转化感受态细胞， 随机挑取 30个单克隆测序。 若 COS细胞中的 p53基因靶位点出现了碱基插入或缺 失突变， 则表明产生了基因突变。
[0042] 2.使用活检穿刺针将步骤 1中制备的 sgRNA表达质粒注射到灵长类动物肝脏门静 脉内， 直至肝脏细胞出现癌变， 获取模型。
[0043] 在该步骤中， 可选用健康的雄性食蟹猴， 5至 8岁， 体重范围 3.2 -6.0kg， 饲养 在中国广西防城港常春生物技术开发有限公司食蟹猴医学应用研究基地， 该研 究基地通过 AAALAC( Assessment And Accreditation Of Laboratory Animal Care)认 证。 在便携式彩超仪 (Terason Co, MA,
USA) 的引导下， 使用活检穿刺针将 sgRNA表达质粒 pX330-p53- sgRNA注射到 食蟹猴肝脏门静脉内。
[0044] 具体步骤如下： 食蟹猴肌肉注射舒眠宁 n注射液 （O. lml/kg） 及注射酚磺乙胺注 射液 （O. lg/只） ， 麻醉成功后食蟹猴取平卧位固定于手术台上， 术区剌毛、 碘 伏消毒、 铺巾。 在 B超引导下， 定位门静脉左支矢状部， 选择安全进针路径， 经 腹部皮肤进针后， 实时观察针尖位置， 将酒精注射疗法针引导到达门静脉左支 矢状部前壁前方， 当有突破感且回抽见血后即可确定针尖成功进入门静脉管腔 。 然后， 向管腔内快速推注 120 ug pX330-p53-sgRNA或对照质粒
pX330-EGFP-sgRNA , 注射体积控制在 400 ^iL内。 紧接着， 再快速推注 0.9%氯化 钠注射液 lml， 超声可见强回声弥散声像， 证实经门静脉注射 CRISPR-Cas质粒系 统成功。 推注完成后， 在超声实时观察下， 将酒精注射疗法针退出体外。 所有 操作完成后， 超声扫查肝脏及肝周， 排除出血及脏器损伤。
[0045] sgRNA表达质粒 pX330-p53-sgRNA肝脏穿刺 45天后， 扩增靶位点并纯化扩增片 段， 对靶位点进行深度测序分析基因编辑情况。 通过深度测序显示， 实验组 6只 食蟹猴， 有 3只食蟹猴在 gRNA靶位点 PAM区域附近检测到了突变， 突变率为 50 % , 突变情况有核酸序列的插入， 也有核酸的缺失。 经过软件分析显示， 插入或 缺失核昔酸的长度分布情况在 lbp-20bp不等， 有 1个碱基的缺失和插入， 也有 20 个碱基的缺失和插入。 对深度测序的结果进行分析， 实验组 6只食蟹猴肝脏组织 p53基因靶位点 Indel的频率最高达到 5.39%。
[0046] 如图 2所示， A为健康食蟹猴代表之一； B为注射对照质粒 pX330-GFP- sgRNA 的食蟹猴代表之一； C为注射 sgRNA表达质粒 pX330-p53-sgRNA食蟹猴代表之一 。 C食蟹猴出现了 p53阳性细胞、 CK19阳性细胞以及 Ki67阳性细胞， 如图中的圆 圈所示。 结果显示， C食蟹猴的肝癌细胞、 胆管上皮细胞中 P53、 Ki67阳性率显 著高于 A食蟹猴和 B食蟹猴。 胆管上皮细胞中 CK19强阳性， 显著高于食蟹猴和 B 食蟹猴。
[0047] 在 sgRNA表达质粒 pX330-p53-sgRNA肝脏穿刺 2个月时， 血清中的肿瘤标志物 A FP、 CA125、 CA19-9明显升高； 肝脏细胞、 胆管上皮细胞出现了向恶性细胞转 变的征象。 这些结果提示肝癌开始形成； 证明了 CRISPR-Cas9系统能够通过 B超 微创介入技术经肝门静脉直接对食蟹猴原位肝脏细胞基因组的 P53基因进行靶向 编辑， 从而引起体细胞 P53抑癌基因缺失突变， 快速诱导肝癌模型的建立。 工业实用性
[0048] 以上所述， 仅为本发明较佳的具体实施方式， 但本发明的保护范围并不局限于 此， 任何熟悉本技术领域的技术人员在本发明揭露的技术范围内， 可轻易想到 的变化或替换， 都应涵盖在本发明的保护范围之内。 因此， 本发明的保护范围 应该以权利要求的保护范围为准。

Claims

权利要求书
[权利要求 1] 一种快速基因编辑构建灵长类动物疾病模型的方法， 用于非诊断或治 疗目的， 其特征在于， 所述方法包括以下步骤：
(a) 使用 gRNA寡核苷酸及 pX330质粒构建 sgRNA表达质粒；
(b) 使用活检穿刺针将所述步骤 (a) 中制备的 sgRNA表达质粒注射 到灵长类动物肝脏门静脉内， 直至肝脏细胞出现癌变， 获取模型。
[权利要求 2] 根据权利要求 1所述的快速基因编辑构建灵长类动物疾病模型的方法
， 其特征在于， 所述步骤 (a) 包括：
(al) 将 pX330质粒用 Bbs I限制性内切酶切割后,用 PCR清洁回收试 剂盒回收；
(a2) 使 gRNA寡核苷酸单链退火形成双链， 连接至 Bbs
I线性化的 pX330质粒， 获得 sgRNA表达质粒。
[权利要求 3] 根据权利要求 1或 2所述的快速基因编辑构建灵长类动物疾病模型的方 法， 其特征在于， 所述步骤 (a) 还包括：
(a3) 对 COS-7细胞使用 DMEM培养基进行细胞培养；
(a4) 取对数生长期的 COS-7细胞， 使用 Lipofectamine 3000转染试剂 转染， 并在转染完成按照基因组提取试剂盒操作步骤提取基因组 DN A；
(a5) 使用 Q5酶对提取的基因组 DNA扩增靶位点， 并进行 T7E1酶切 检测及 TA克隆测序， 在确认基因组 DNA中 P53基因靶位点序列产生基 因突变后执彳了步骤 (b) 。
[权利要求 4] 根据权利要求 3所述的快速基因编辑构建灵长类动物疾病模型的方法 ， 其特征在于： 所述步骤 (a4) 包括： 取对数生长期的 COS-7细胞铺 六孔板， 每孔细胞 1.5 x 10 6， 当细胞密度达到 70%时，
使用 Lipofectamine 3000转染试剂转染， 每个孔转染 sgRNA表达质粒的 量控制在 2.5-3.0 ， 转染后 48小时收细胞， 按照基因组提取试剂盒操 作步骤提取基因组 DNA。
[权利要求 5] 根据权利要求 1所述的快速基因编辑构建灵长类动物疾病模型的方法 ， 其特征在于： 所述 gRNA寡核苷酸包括碱基序列 5’ -
CAATTCTGCCCT CACAGCTC - 3，。
[权利要求 6] 根据权利要求 1所述的快速基因编辑构建灵长类动物疾病模型的方法 ， 其特征在于： 所述步骤 (b) 包括：
(bl) 在 B超引导下， 定位门静脉左支矢状部， 选择安全进针路径， 经腹部皮肤进针后， 实时观察针尖位置， 将酒精注射疗法针引导到达 门静脉左支矢状部前壁前方， 当有突破感且回抽见血后即可确定针尖 成功进入门静脉管腔；
(b2) 向管腔内快速推注 120 ug pX330-p53-sgRNA或对照质粒 pX330-EGFP-sgRNA, 注射体积控制在 400 pL内；
(b3) 快速推注 0.9%氯化钠注射液 lml， 超声可见强回声弥散声像， 证实经门静脉注射 CRISPR-Cas质粒系统成功；
(b4) 推注完成后， 在超声实时观察下， 将酒精注射疗法针退出体外
(b5) 所有操作完成后， 超声扫查肝脏及肝周， 排除出血及脏器损伤
[权利要求 7] 根据权利要求 6所述的快速基因编辑构建灵长类动物疾病模型的方法 ， 其特征在于： 所述步骤 (b5) 之后还包括： 45天后， 扩增靶位点并 纯化扩增片段， 对靶位点进行深度测序分析基因编辑情况。
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