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నైరూప్య

A new approach to introduce specific point mutations in the gene coding for dystrophin: PRIME editing technology.

Cédric Happi Mbakam

Mutations in the dystrophin gene lead to neuromuscular disorderssuch as Duchenne Muscular Dystrophy which is a lethalX-linked hereditary disease with the prevalence of 19.8 per 100000 males’ birth. Currently available clinical therapies with corticosteroidsor with morpholino antisense oligomer injections providelimited phenotypic improvement. Our study aimed to measurethe PRIME editing technology efficiency. This technologyuses a PRIME editor plasmid (PE2 or PE3) coding for a Moloneymurine leukemia virus reverse transcriptase fused with the Cas9H840A nickase, and a plasmid coding for a pegRNA containinga primer binding sites (PBS) and a reverse transcriptase template(RTT). It permits specific nucleotide substitutions, deletions orinsertions in the genome. We designed different pegRNAs targetingseveral hDMD exons (9, 20, 35,43, 51, 55, and 61) to introducea STOP codon by modifying a single nucleotide. HEK293Tcells were harvested from DMEM culture media three days afterbeing simultaneously transfected with the PE2 and pegRNA. Exonswere PCR amplified and sequenced using the Sanger method.Results were analysed using the EditR program to estimate the editingpercentage. We confirmed that PRIME editing permits thespecific C to T and G to T substitutions in the DMD gene withan editing efficiency between 6 to 11 % (PE2) and 21% (PE3).Repeated transfections 6 days after the first one showed up to15 % (PE2) edition in exons 9 and 35. An additional mutationin PAM sequence (exon 35) improved a PE2 result to 38% for asingle transfection. Thus, PRIME editing permits the specific substitutionsin the DMD gene and might be used to correct pointmutations in the DMD gene to lead to dystrophin expression.