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Production of virus-like particles with AsCas12a nuclease and CMV-driven crRNA for mammalian genome editing
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DOI：10.3389/fgeed.2026.1735339.png)
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Abstract
En 中文
CRISPR/Cas genome editing tools represent a promising technology for biomedicine with significant therapeutic potential for numerous human diseases. However; efficient delivery of these tools into primary cells; particularly in the form of ribonucleoprotein (RNP) complexes; remains a critical bottleneck that limits clinical translation. Virus-like particles (VLPs) derived from human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) have emerged as promising delivery vehicles for RNP complexes; yet their activity is limited by suboptimal nuclease and guide RNA packaging. Previously; we generated NanoMEDIC VLPs incorporating the AsCas12a nuclease with CMV-driven crRNA; which demonstrated substantially enhanced editing efficiency over SpCas9-VLPs with U6-driven gRNA. Here; we describe a detailed protocol for a small-scale production of AsCas12a-VLPs using three distinct transfection methods [cationic lipids; polyethyleneimine (PEI); and calcium-phosphate] and a large-scale production of VLPs using calcium-phosphate transfection. We show that both production scales yield comparable nuclease loading into VLPs and similar editing efficiencies; reaching up to 60% of CXCR4 knockout in Jurkat T cells.
Keywords:
virus-like particles
genome editing
ribonucleoprotein complexes
CRISPR/Cas delivery
AsCas12a
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