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Chronic hepatitis B infection remains incurable due to the stable presence of various forms of hepatitis B virus (HBV) genome, especially the HBV covalently closed circular DNA (cccDNA). The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) technology provides a new opportunity to potentially cure the HBV infection. However, the efficiency and specificity remain unsatisfactory, especially for nonviral CRISPR/Cas9 delivery. To tackle these, a liver‐specific CRISPR/Cas9 magnetic nanosystem FMNPpAG333/sgXPP is constructed based on fluorinated polyethylenimine‐coated magnetic nanoparticles and liver‐specific ApoE.HCR.hAAT promoter‐driven Cas9‐T2A‐EGFP plasmid with dual sgRNAs. The elaborate system enables magnetic field‐induced spatially specific distribution and hepatocyte‐specific promoter‐driven liver‐specific gene editing. Moreover, this CRISPR/Cas9 magnetic nanosystem is designed to disrupt the two conserved sites in X opening reading frame and Pol opening reading frame of the HBV genome, thereby significantly inactivating the HBV genome without showing off‐target effects. Treatment with FMNPpAG333/sgXPP for 7 days reduces serum HBsAg levels by 76% with a total editing efficiency of ≈20% in the two conserved sites. Collectively, this study demonstrates spatiotemporal liver genome editing as well as the feasibility of applying a nonviral CRISPR/Cas9 vector for HBV treatment, which may open up a new application for CRISPR therapeutics. [ABSTRACT FROM AUTHOR]

Liver disease, particularly viral hepatitis and hepatocellular carcinoma (HCC), is a global healthcare burden and leads to more than 2 million deaths per year worldwide. Despite some success in diagnosis and vaccine development, there are still unmet needs to improve diagnostics and therapeutics for viral hepatitis and HCC. The emerging clustered regularly interspaced short palindromic repeat/associated proteins (CRISPR/Cas) technology may open up a unique avenue to tackle these two diseases at the genetic level in a precise manner. Especially, liver is a more accessible organ over others from the delivery point of view, and many advanced strategies applied for nanotheranostics can be adapted in CRISPR‐mediated diagnostics or liver gene editing. In this review, the focus is on these two aspects of viral hepatitis and HCC applications. An overview on CRISPR editor development and current progress in clinical trials is first given, followed by highlighting the recent advances integrating the merits of gene editing and nanotheranostics. The promising systems that are used in other applications but may hold potentials in liver gene editing are also discussed. This review concludes with the perspectives on rationally designing the next‐generation CRISPR approaches and improving the editing performance. [ABSTRACT FROM AUTHOR]

Abstract: CRISPR/Cas9 technology enables targeted gene editing; yet, the efficiency and specificity remain unsatisfactory, particularly for the nonvirally delivered, plasmid‐based CRISPR/Cas9 system. To tackle this, a self‐assembled micelle is developed and evaluated for human papillomavirus (HPV) E7 oncogene disruption. The optimized micelle enables effective delivery of Cas9 plasmid with a transient transgene expression profile, benefiting the specificity of Cas9 recognition. Furthermore, the feasibility of using the micelle is explored for another nucleic acid‐guided nuclease system, Natronobacterium gregoryi Argonaute (NgAgo). Both systems are tested in vitro and in vivo to evaluate their therapeutic potential. Cas9‐mediated E7 knockout leads to significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo, while NgAgo does not show significant E7 inhibition on the xenograft mouse model. Collectively, this micelle represents an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine‐based therapeutics. [ABSTRACT FROM AUTHOR]