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SCIENTIA SINICA Chimica, Volume 49, Issue 5: 752-765(2019) https://doi.org/10.1360/N032018-00233

Research progress on non-viral vectors of CRISPR/Cas9 system

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  • ReceivedOct 29, 2018
  • AcceptedDec 12, 2018
  • PublishedFeb 27, 2019

Abstract

In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system has become one of the most powerful technologies in biomedical research and shown great potentials in the establishment of disease models as well as the development of new treatments. However, efficient delivery of CRISPR/Cas9 systems to target organs and cells remains challenging. With the rapid development of non-viral vectors, nanocarriers based on liposomes, polymers, and inorganic nanoparticles have shown great potentials for the delivery of CRISPR/Cas9 systems. In this review, we first summarize the potential of the CRISPR/Cas9 system in disease modeling and disease treatment, as well as the limitations. The advantages and disadvantages of delivering the CRISPR/Cas9 system in the form of plasmid DNA, mRNA or protein are then analyzed and the non-viral vectors that have emerged are reviewed. Finally, we summarize the key barriers currently faced by non-viral vectors in the delivery of CRISPR/Cas9 systems and propose strategies that are expected to overcome them.


Funded by

国家重点研发专项“纳米科技”(2018YFA0209700)

国家自然科学基金(51673100)

天津市自然科学基金(18JCQNJC03600)


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  • Figure 1

    Different strategies for delivering CRISPR/Cas9 system. (a) Delivery of pDNA encoding Cas9 protein and sgRNA. (b) Delivery of Cas9 mRNA and sgRNA. (c) Delivery of RNP of Cas9 protein and sgRNA (color online).

  • Figure 2

    Non-viral vector for delivery of pDNA encoding the CRISPR/Cas9 system [77,78,83,84] (color online).

  • Figure 3

    Non-viral vector for delivery of mRNA encoding the Cas9 protein [93,95,96] (color online).

  • Figure 4

    Non-viral vector for delivery of Cas9 protein/sgRNA and functional engineering of Cas9 protein [81,87,88,92] (color online).

  • Table 1   Physical methods for delivering the CRISPR/Cas9 system

    递送方法

    主要递送形式

    优点

    缺点

    参考文献

    显微注射法

    pDNA, mRNA, RNP

    确保靶标到目的细胞上

    费时费力, 操作困难, 仅限于体外应用

    [63~65]

    电导转入法

    pDNA, mRNA, RNP

    操作简单, 适用于递送Cas9蛋白

    瞬间电压会引起细胞的非正常凋亡并丧失细胞活性

    [70,71]

    尾静脉高压注射法

    pDNA, RNP

    价格低廉, 操作简单

    非特异性; 对肝脏肾脏有明显损伤

    [72]

  • Table 2   Non-viral vectors for delivering the CRISPR/Cas9 system

    递送载体

    主要递送形式

    优点

    缺点

    参考文献

    聚乙烯亚胺

    pDNA, mRNA, RNP

    较高的电荷密度和优异的pH缓冲能力

    高分子量PEI具有较高的毒性, 且体内应用时容易被清除

    [77~81]

    壳聚糖

    pDNA, mRNA

    易修饰, 生物毒性低

    在递送大分子量pDNA时会由于太过紧密的缠绕而影响转染效率

    [82,83]

    聚氨基酸

    pDNA

    聚合度高度可控, 易于化学修饰, 体内可生物降解

    伯胺的电荷密度相对较低会影响溶酶体逃逸及转染效率

    [84~86]

    DNA纳米链

    RNP

    可大幅度保留Cas9蛋白的生物活性, 并实现高效的目的基因剪切

    需要对DNA球体进行分别设计, 步骤繁琐, 价格昂贵

    [87]

    金纳米颗粒

    RNP, pDNA

    这种基于膜融合的递送方法, 递送效率极高

    在体内可能会引起非特异性的炎症反应及免疫效应

    [88~92]

    脂质体

    mRNA, pDNA, RNP

    商业产品, 可直接购买, 操作简单

    溶酶体逃逸效率低, 缺少细胞靶向性

    [93~96]

    无机纳米粒子

    RNP, pDNA

    递送效率高

    合成过程复杂, 不易被代谢

    [97,98]

    细胞穿膜肽

    pDNA, mRNA, RNP

    安全, 粒径较小

    需要进一步化学修饰

    [99,100]

    正电性蛋白

    pDNA, RNP

    生物相容性较好, 粒径较小

    效率较低, 体内递送较为困难

    [101,102]

    外泌体

    pDNA

    生物相容性好

    制备过程复杂

    [103]

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