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SCIENCE CHINA Life Sciences, Volume 60, Issue 5: 458-467(2017) https://doi.org/10.1007/s11427-017-9033-0

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field

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  • ReceivedJan 16, 2017
  • AcceptedMar 16, 2017
  • PublishedMay 2, 2017

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs (sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies. Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.


Funded by

National Natural and Scientific Foundation of China(81602699 to Zhi-Yao He,81502677 to Ke Men,81402302 to Yang Yang)

National High Technology Research and Development Program of China(2015AA020309 to Zhi-Yao He)

China Postdoctoral Science Foundation Funded Project(2015M570791 to Zhi-Yao He)


Acknowledgment

This work was supported by the National Natural and Scientific Foundation of China (81602699 to Zhi-Yao He, 81502677 to Ke Men, 81402302 to Yang Yang), the National High Technology Research and Development Program of China (2015AA020309 to Zhi-Yao He), and the China Postdoctoral Science Foundation Funded Project (2015M570791 to Zhi-Yao He).


Interest statement

The author(s) declare that they have no conflict of interest.


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  • Figure 1

    Schematic diagram of CRISPR-Cas9-mediated genome editing. Cas9 is guided by an sgRNA to induce a double-strand DNA break (DSB) at a desired genomic locus. The DSB can be repaired by NHEJ causing random insertion or deletion (indel) mutations or by HDR using a donor DNA template, enabling the introduction of desired sequence changes for precise genome editing purposes.

  • Figure 2

    Delivery vectors for CRISPR-Cas9 systems. Human codon-optimized Cas9 and sgRNA sequences were packaged into a viral vector (e.g., adenovirus, rAAV, lentivirus) for genome editing. Cas9 protein, mRNA of Cas9 and sgRNA, or a plasmid encoding Cas9 and sgRNA was incorporated into a nanoparticle to formulate a nano-Cas9 complex for non-viral delivery.

  • Table 1   Non-viral and viral vectors for CRISPR-Cas9 system and their applications in the biomedical field

    Delivery methods

    Advantages

    Disadvantages

    Applications

    Microinjection

    High efficiency in vitro

    Low-throughput

    Genome editing for oocytes or embryos; generation of model animals

    Electroporation

    High transfection efficiency in vitro

    Cytotoxicity, difficult for in vivo use

    Genome editing for various cell types in vitro

    Hydrodynamic injection

    Feasible for in vivo gene editing in small animals

    Low efficiency, difficult for clinical use

    Gene function study in vivo

    CPP

    Low off-target effects

    Low efficiency, immunogenicity, difficult for in vivo use

    Genome editing for cells in vitro

    Cationic vectors

    Easy to produce, large packaging capacity

    Low efficiency

    Genome editing for various cell types in vitro; gene therapy for cancer, HBV, genetic diseases, etc.

    Retrovirus

    High efficiency in vivo, integrating target gene into host cell genome

    Insertional mutagenesis, oncogene activation

    Gene therapy for cancer, genetic diseases, etc.

    Lentivirus

    High efficiency, high throughput in vitro and in vivo

    Prone to rearrangements of cargo genes, liable to transgene silencing

    Genomic screen and gene function study in vitro and in vivo

    Adenovirus

    High efficiency in vivo, high packaging capacity

    Immunoreactivity, difficult to produce in large scale

    Gene therapy for genetic diseases

    AAV

    High efficiency in vivo, non-pathogenic

    Limited packaging capacity, high cost

    Gene therapy for various genetic diseases

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