Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) systems have emerged as a robust and versatile genome editing platform for gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. However, the insufficient transfection and off-target risks have seriously hampered the potential biomedical applications of CRISPR/Cas9 technology. Herein, we review the recent progress towards CRISPR/Cas9 system delivery based on viral and non-viral vectors. We summarize the CRISPR/Cas9-inspired clinical trials and analyze the CRISPR/Cas9 delivery technology applied in the trials. The rational-designed non-viral vectors for delivering three typical forms of CRISPR/Cas9 system, including plasmid DNA (pDNA), mRNA, and ribonucleoprotein (RNP, Cas9 protein complexed with gRNA) were highlighted in this review. The vector-derived strategies to tackle the off-target concerns were further discussed. Moreover, we consider the challenges and prospects to realize the clinical potential of CRISPR/Cas9-based genome editing.
Highlights
The Cas9 ribonucleoprotein (RNP)-based clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system could give the swiftest gene editing via a skip of the expression of protein and single-guide RNA (sgRNA) in cells compared with the plasmid and RNA forms of CRISPR/Cas9
CRISPR/Cas9 holds tremendous potential as a therapeutic for diverse diseases related to genetic disorders such as β-thalassaemia, tyrosinemia, and cancers
The CRISPR/Cas9-based therapies have been evaluated in clinical trials
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The CRISPR/Cas system possesses two crucial components, including gRNA and Cas protein, to enable effective genome editing in eukaryotic cells. Upon the guidance of sgRNA, Cas protein can target any genomic locus via base pairing and induce a DSB. Prior to the CRISPR/Cas system, DNA recognition domain-containing endonucleases including meganucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effectors (TALENS) were the dominating tools for gene editing [8]. The earliest meganucleases possess varying DNA sequence specificity, which was enabled by protein engineering of recognition sites. Subsequent ZFNs and TALENs further unlock the potential of genome engineering They simultaneously consisted of a non-specific FokI nuclease domain and tailor-made DNA recognition domains. The CRISPR/Cas system shares tremendous promise for various applications including gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. This review highlights rationally designed viral and non-viral vectors, and provides prospects for future CRISPR-Cas research
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