Lipofection- and lentivirus-mediated protocols for CRISPR-Cas9 delivery in mouse-passaged primary human hepatocytes (mpPHH) are reported, a system that enables PHH expansion in liver-humanized mice and enables scalable genetic manipulation of mpPHH, opening new avenues for HBV research and liver disease modeling.
Abstract
More than two million deaths annually are attributed to liver-related conditions, making primary human hepatocytes (PHH) an invaluable in vitro model for studying liver pathophysiology and the molecular mechanisms underlying hepatic diseases. However, because PHH do not proliferate in culture, CRISPR gene editing has been highly inefficient. Here, we report lipofection- and lentivirus-mediated protocols for CRISPR-Cas9 delivery in mouse-passaged primary human hepatocytes (mpPHH), a system that enables PHH expansion in liver-humanized mice. We achieve robust gene editing efficiencies exceeding 90% in mpPHH while maintaining cell viability. We demonstrate the utility of these protocols by disrupting CYP3A4 to impair xenobiotic metabolism and by showing that edited mpPHH efficiently engraft and expand in mice, generating liver-humanized animals. We establish the feasibility of arrayed CRISPR screening in mpPHH using an 85-gene screen to identify host factors influencing hepatitis B virus (HBV) infection, and validate key findings in humanized mice by targeting the HBV entry receptor SLC10A1 (NTCP), which reduced viral infection in vivo. Our methodology enables scalable genetic manipulation of mpPHH, opening new avenues for HBV research and liver disease modeling.
The first pooled, genome-wide CRISPR-Cas9 knockout (KO) screen with authentic HBV infection is performed and these results are integrated with two orthogonal pooled screens to identify host factors to expand the catalog of HBV host factors and provide a scalable framework for host target discovery.
C. A. Freije, Georgios Dangas, Antonis Athanasiadis et al.· bioRxiv· 0 citations
A virus-like particle (VLP)-based toolkit that delivers diverse CRISPR editing modalities to human monocytes, macrophages and dendritic cells with high efficiency while preserving viability and innate immune responsiveness is presented.
Hyuncheol Jung, Pascal Devant, Carter Ching et al.· Nature Biotechnology· 0 citations
RAAV integration patterns in primary human hepatocytes xenografted into FRG mouse livers and in hepatocytes from cynomolgus macaques following systemic rAAV administration support a low oncogenic risk profile for the evaluated vector while reinforcing the value of direct human liver integration analyses to refine risk assessment and guide the development of safer gene therapy platforms.
S. Scott, C. Hallwirth, Natsuki Sasaki et al.· Molecular Therapy· 0 citations
Human induced pluripotent stem cells (hiPSCs) represent a powerful platform for disease modeling, especially in monogenic diseases as they preserve the donor’s genetic background while enabling directed differentiation into disease-relevant cell types. This makes them highly suitable for studying disease mechanisms in a patient-specific and physiologically relevant context. Although CRISPR/Cas9 is widely applied for genome editing, precise correction of pathogenic variants in hiPSCs remains challenging due to the lack of standardized CRISPR component selection and experimental design. Here, we describe an optimized CRISPR-based strategy for correcting a heterozygous HNF1A frameshift mutation (c.235_236insG; p.Glu79Glyfs*16) in HNF1A-MODY patient-derived hiPSCs. Using electroporation, we efficiently delivered CRISPR components, including a ribonucleoprotein complex of Cas9 and single-guide RNA, along with a single-stranded oligodeoxynucleotide repair template. Corrected hiPSC lines were validated for pluripotency, absence of exogenous reprogramming factors, and off-target effects. Additionally, we discuss key technical challenges encountered during the editing process and provide practical recommendations that may improve the generation of mutation-corrected hiPSC lines. These guidelines could serve as a useful reference for researchers employing CRISPR-based strategies for generation of reliable disease modelling tools.
D. Skoczek, Jerzy Hohendorff, Maciej T. Małecki et al.· Human Genetics· 0 citations
The results showed the potential of a combinatorial AAV library for model validation and revealed the human microliver platform-PEG as a reliable system for the development of AAV therapeutics.
Carmen Unzu, Amanda X. Chen, Liliana Mancio-Silva et al.· bioRxiv· 0 citations
