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 editing [1]. 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 delivery [2]. A previous study has combined lipid nanoparticle and AAV vector to deliver CRISPR/Cas9 system components [3]. 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 hypercholesterolemia [5]. To the best of our knowledge, this is the first report on non-viral delivery of CRISPR/Cas9 system components (mRNA+ sgR-NA) in adult animals.

Previously, we reported the development and optimization of TT3 LLNs (Supplementary information, Figure S1A), which are capable of efficiently delivering mRNA both in vitro and in vivo [4]. To optimize the LLN formulation for Cas9 mRNA delivery, we designed 27 formulas of TT3 LLNs (Supplementary information, Table S1) and examined their ability to delivery Cas9 mRNA into HEK293T cells stably expressing EGFP and an sgRNA targeting EGFP [3]. Forty-eight hours after the delivery, we quantified the percentage of EGFP-positive cells by fluorescence-activated cell sorting (FACS) analysis. Formula 3 showed the highest delivery efficiency, reducing the percentage of EGFP-positive cells by over 50%