Targeted gene removal by SpCas9 transduced by a protein-RNA complex transduction system NanoMEDIC

Abstract

The CRISPR-Cas9 system offers powerful genome editing capabilities, but off-target effects remain a key limitation. Direct delivery of Cas9 protein complexed with guide RNA (gRNA) can mitigate these effects by limiting exposure time. We previously developed NanoMEDIC, a virus-like particle system shown to deliver Cas9/gRNA ribonucleoprotein (RNP) complexes both in vitro and in vivo. In the present study, we compared NanoMEDIC-mediated delivery with plasmid-based transfection using a gene excision reporter in the human embryonic kidney-derived cell line 293FT and the human glioblastoma-derived cell line NP-2. In this model, the DNA sequence targeted by Cas9/gRNA was derived from a human oncogenic retrovirus, human T-cell leukemia virus type I (HTLV-1). NanoMEDIC achieved efficient gene removal-editing with significantly lower Cas9 input per cell regardless of whether the target DNA was transiently transfected or integrated into chromosomal DNA. Sequencing of edited loci revealed that NanoMEDIC-mediated Cas9/gRNA delivery produced 58.3 – 87.5 % removal-edited DNA without insertions or deletions (indels), compared with 8.3 – 29.4 % using plasmid transfection. These data suggest a high editing precision of NanoMEDIC-mediated Cas9/gRNA delivery. This likely reflects the transient presence of Cas9, which enables rapid repair through non-homologous end joining (NHEJ) without prolonged nuclease activity. Collectively, our findings highlight the potential of NanoMEDIC for applications requiring precise, indel-free genome modifications and support its use as a safer alternative to DNA-based delivery or systems involving sustained Cas9 expression.