The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has revolutionized biomedical research and facilitated the development of new therapies based on genome editing1. A major roadblock to achieve the therapeutic potential of the CRISPR/Cas system is the lack of a safe and effectiving in vivo delivery method. Adeno-associated virus (AAV)-assisted delivery of the CRISPR/Cas9 system has shown gene targeting efficacy in vivo, however, the long persistence and immunogenicity of AAV in the host prevent the wide therapeutic application of AAV-based CRISPR/Cas9 delivery2. A previous study has combined lipid nanoparticle and AAV vector to deliver CRISPR/Cas9 system components3. Here we show that by using an optimized formula of our newly developed lipid-like nanoparticles (LLNs)4, we are able to effectively deliver Cas9 mRNA and single-guide RNA (sgRNA) to the liver and achieve in vivo targeting of HBV DNA and the proprotein convertase subtilisin/kexin type 9 (pcsk9) gene, a therapeutic target for treating hypercholesterolemia5. To the best of our knowledge, this is the first report on non-viral delivery of CRISPR/Cas9 system components (mRNA + sgRNA) in adult animals.

HBV covalently closed circular DNA (cccDNA), the major form of the viral genome in infected hepatocytes, represents an intractable barrier to eradication of the virus and is resistant to the currently available therapies6. Several previous studies have explored the potential of the CRISPR/Cas9 system to target HBV cccDNA in mice by hydrodynamic injection of plasmid DNA7,8,9,10,11; however, therapeutically relevant in vivo delivery methods are still lacking12. To systematically identify potential sgRNAs targeting HBV DNA, we used the CRISPR DESIGN tool (http://crispr.mit.edu) to design potential sgRNAs. To achieve a broad-spectrum antiviral activity, we selected 9 sgRNAs that target regions with the highest levels of conservation among HBV strains (Supplementary information, Figure S1E)13. We tested the sgRNAs using an in vitro transfection assay in HepAD38 cells, a hepatocyte line with an integrated HBV genome. We found that when each combined with Cas9, several sgRNAs efficiently diminished HBV protein production as measured by ELISA of HBV surface antigen (HBsAg) (Figure 1B). We also performed T7E1 assays in a cell model that produces episomal HBV cccDNA14, and found that together with Cas9, all sgRNAs except for B9 induced indels in the HBV DNA (Figure 1C). We selected the most potent sgRNA B5 (sgB5) based on its performance in ELISA for further analysis. We found that sgB5 delivered by TT3 LLNs via tail vein injection can be detected in the mouse liver by qRT-PCR and its level decreased significantly 4-8 h post injection (Figure 1D).

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