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Life sciences · Journal article
Acs Applied Materials & Interfaces · October 16, 2026
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Abstract Delivery of CRISPR-Cas9 ribonucleoprotein (RNP) using synthetic materials can bypass the host translational machinery and limit the duration of genomic exposure, minimizing the risk of off-target mutations. Despite their advantages, lipid nanoparticle (LNP) platforms for CRISPR-Cas9 RNP delivery that achieve highly efficient editing at low doses remain limited. In the present study, we developed a library of ionizable lipids (ILs) and corresponding LNPs to enhance the delivery and editing efficiencies of CRISPR-Cas9 RNP complexes. Following comprehensive in vitro screening and formulation optimization, an optimal LNP candidate containing the C12A9 IL (denoted as LNPC12A9) was identified that achieved ∼90% EGFP disruption in HEK293T-EGFP cells at an RNP concentration of 30 nM and ∼60% at 15 nM. The editing performance was consistently observed at an RNP concentration of 15 nM when editing the Gcgr and HPRT1 genes across multiple hepatic cell lines. Moreover, coencapsulation of RNP and a single-stranded oligodeoxynucleotide (ssODN) template for homology-directed repair (HDR) within LNPC12A9 induced an HDR rate of 37.8%. Systemic administration of LNPC12A9 into C57BL/6J mice led to liver tropism and a total hepatic Pcsk9 gene disruption rate of 1.39%. In conclusion, this study provides insights into the construction of an LNP platform to improve the delivery efficiency of CRISPR-Cas9 RNP, thereby facilitating the development of LNPs for safe, precise, and tissue-specific Cas9 RNP-based genome editing therapies.