Comparative analysis of single and dual sgRNA CRISPR/Cas9 ribonucleoprotein strategies for efficient Park7 gene editing in primary mouse embryonic fibroblasts.

BACKGROUNDParkinson's disease (PD) is a progressive neurological condition that predominantly affects dopaminergic neurons in the substantia nigra. Mutations or deletions in the Park7 gene may cause a rare autosomal recessive form of early-onset PD. Generating reliable cellular models with specific Park7 disruption is crucial for elucidating the molecular mechanisms underlying PD pathogenesis.

METHODS AND RESULTSThis study developed a plasmid-free CRISPR/Cas9 genome-editing method for targeting the Park7 gene in primary mouse embryonic fibroblasts (MEFs) using single- and dual-sgRNA-mediated ribonucleoprotein (RNP) system. Gene modification through the single sgRNA strategy was confirmed by the detection of indels by T7 endonuclease assay, whereas gene editing by the dual sgRNA strategy was validated through detection of large deletions by junctional PCR and Sanger sequencing. Deep amplicon sequencing showed an overall editing frequency of 50.68% and 24.87% for single-sgRNA and dual-sgRNA editing strategies, respectively. While single-sgRNA editing predominantly generated small insertions and deletions, large deletions constituted the predominant editing outcome in the dual-sgRNA group, accounting for 22.23% of the total reads. Further, Western blot analysis demonstrated a 12.03 ± 7.6% reduction in PARK7 protein expression following single sgRNA transfection, whereas dual sgRNA transfection showed a significant reduction of 54.89 ± 9.1% (p < 0.0001).

CONCLUSIONThis study demonstrates a plasmid-free CRISPR/Cas9 RNP-based approach for targeted Park7 gene editing in primary MEFs. The results underline the importance of considering both the outcomes of genomic editing and the expression of functional protein when evaluating CRISPR/Cas9 approaches. This established approach sets the basis for the development of Park7-edited cellular models to study molecular mechanisms associated with PD and the subsequent development of disease-relevant models.