AbstractThe broad application of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing tools is hindered by challenges in the efficient delivery of its two components into specific cells and intracytoplasmic release. Herein, a novel copolymer for delivery of Cas9‐mRNA/ single‐guide RNA (Cas9‐mRNA/sgRNA) in vitro and vivo, using carboxylesterase‐responsive cationic triadic copolymeric nanoparticles targeted proprotein convertase subtilisin/kexin type 9 (PCSK9) for hyperlipidemia amelioration is reported. A dimethyl biguanide derivative is designed and synthesized to form cationic block, and copolymerization onto prepolymer with propyl methacrylate, to fabricate a triadic copolymer mPEG‐b‐P(Met/n‐PMA). The copolymer can self‐assemble with Cas9‐mRNA/sgRNA, indicating the excellent potential of nanoparticles to form a delivery carrier. This vehicle can efficiently release RNA in response to the hepatocytes carboxylesterase for genome editing. It was demonstrated that the mPEG‐b‐P(Met/n‐PMA)/Cas9 mRNA/sgRNA nanoparticles effectively accumulated in hepatocytes, lead to the inhibition of PCSK9, and lowered the levels of Low‐density lipoprotein cholesterol and total cholesterol in mouse serum down 20% of nontreatment. Interestingly, the nanoparticles even enable multiple functions in the regulation of blood glucose and weight. This study establishes a novel method to achieve complex CRISPR components stable loading, safe delivery, and fixed‐point release, which expand the application of CRISPR delivery systems.