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化学进展 2020, Vol. 32 Issue (1): 5-13 DOI: 10.7536/PC191218 前一篇   后一篇

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基于CRISPR的生物分析化学技术

李悦, 李景虹**()   

  1. 1. 清华大学化学系 生命有机磷化学及化学生物学教育部重点实验室 北京 100084
  • 收稿日期:2019-12-22 出版日期:2020-01-15 发布日期:2020-01-08
  • 通讯作者: 李景虹
  • 基金资助:
    国家自然科学基金项目资助(21621003)
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CRISPR Bioanalytical Chemistry Technology

Yue Li, Jinghong Li**()   

  1. 1. Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology(Ministry of Education), Tsinghua University, Beijing 100084, China
  • Received:2019-12-22 Online:2020-01-15 Published:2020-01-08
  • Contact: Jinghong Li
  • About author:
    ** E-mail:
  • Supported by:
    National Natural Science Foundation of China(21621003)

CRISPR(Clustered regularly interspaced short palindromic repeats)技术是一种革命性的基因编辑和调控工具,问世之后迅速成为了生物医学领域的前沿热点,广泛用于基因功能研究和治疗。CRISPR具有优异的序列识别性质,核酸切割能力,并且易于编程设计改造,在生物分析化学领域展示出独特的魅力,在病毒检测、临床诊断和单细胞成像分析等方面都取得了突破性进展。目前基于CRISPR技术的检测方法种类繁多,本文综述了CRISPR-Cas分析检测方法的研究进展,并且展望了该技术的发展趋势。

关键词： CRISPR, 生物分析, 临床检测, 生物传感器, 单细胞成像

CRISPR (Clustered regularly interspaced short palindromic repeats) technology is a revolutionary gene editing tool developed in recent years, which has quickly become a cutting-edge technology in the field of biology and medicine and widely used in gene function research and gene therapy. In addition to excellent gene editing capabilities, various new functions are also being continuously created with the efforts of scientists from different fields. CRISPR has excellent sequence recognition properties, nucleic acid cleavage ability, and is easy to program and design. Therefore, it exhibits unique charm in the field of bioanalytical chemistry, and has made breakthrough progress in virus detection, clinical diagnosis, and single cell imaging analysis. At present, there are many types of detection methods using CRISPR technology. Therefore, we summarize and classify the current research status of CRISPR analysis and detection methods, and look forward to its development trend.

Key words: CRISPR, bioanalysis, clinical detection, biosensor, single cell imaging
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图1 CRISPR-Cas核酸酶活性概述:(a)在gRNA引导下,Cas9蛋白的RuvC和HNH核酸酶结构域可以靶向剪切DNA双链。在Cas9基础上对核酸酶结构域进行突变,产生具有切口酶活性的Cas9 nickase HNH+和Cas9 nickase RuvC+,以及没有剪切活性的dCas9。(b)Cas12a识别剪切特异序列的ssDNA或dsDNA,并激活非特异性的DNA酶活性。(c)Cas13a识别剪切特异序列的ssRNA,并激活非特异性的RNA酶活性
Fig. 1 The nuclease activity of CRISPR-Cas protein: (a) Under the guidance of gRNA, the nuclease domains of Cas9 protein, RuvC and HNH, would target DNA double strands. Mutating of the nuclease domain on the basis of Cas9 can produce Cas9 nickase HNH+ and Cas9 nickase RuvC+, and deactivated dCas9. (b) Cas12a recognizes and cleaves ssDNA or dsDNA with specific sequences and activates non-specific DNase activity. (C) Cas13a recognizes and cleaves ssRNA with specific sequences and activates non-specific RNase activity
图2 基于CRISPR-Cas9的核酸检测技术:(a)在纸基试纸上利用Cas9的序列识别能力实现ZIKV病毒检测分型[37];(b)CRISPR-Cas9引发的切口酶介导的链置换扩增法(CRISDA)检测人和转基因大豆基因[40];(c)用一对荧光素酶修饰的dCas9对病原菌特征核酸序列检测[42]
Fig. 2 CRISPR/Cas9 based nucleic acid detection: (a) ZIKV virus detection and genotyping on paper test strips[37]; (b) detection of human and transgenic soybean genes using CRISPR-Cas9-triggered nicking endonuclease mediated strand displacement amplification method (CRISDA)[40]; (c) detection of characteristic nucleic acid sequences of pathogenic bacteria with a pair of luciferase-modified dCas9[42]
图3 Cas12a或Cas13a核酸检测技术原理
Fig. 3 Workflow of Cas12a or Cas13a nucleic acid detection
图4 (a)Cas12a和别构转录因子介导的小分子检测技术(CaT-SMelor)[56];(b)Cas12a和适配体介导的小分子检测技术[57];(c)Cas12a的智能响应水凝胶材料[58]
Fig. 4 (a) CRISPR-Cas12a- and aTF-mediated small molecule detector (CaT-SMelor)[56]. (b) CRISPR-Cas12a- and aptamer-mediated small molecule detector[57]. (c) Cas12a-responsive smart materials[58]
图5 CRISPR成像技术原理:(a)dCas9蛋白上修饰荧光蛋白;(b)gRNA骨架中插入适配体序列,由噬菌体衣壳修饰荧光蛋白,适配体序列和噬菌体衣壳相结合;(c)在dCas9上修饰重复的抗原表位决定簇,将荧光蛋白连接在抗体上,抗体和抗原表位决定簇相结合;(d)gRNA骨架上可以修饰重复的适配体序列,招募多个噬菌体衣壳修饰的荧光蛋白
Fig. 5 Illustration of CRISPR imaging technology: (a) dCas9 protein was fused with fluorescent protein; (b) The aptamer sequence was inserted into gRNA backbone and bound to a fluorescent protein fused to the phage capsid. (c) Repeated epitope determinants were fused on dCas9 and bound to antibodies fused to fluorescent proteins; (d) Repeated aptamers were inserted to the gRNA backbone to recruit multiple fluorescent proteins fused to the phage capsid
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摘要

基于CRISPR的生物分析化学技术