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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 lethal X-linkedhereditary disease with the prevalence of 19.8 per 100 000 males’birth. Currently available clinical therapies with corticosteroids orwith morpholino antisense oligomer injections provide limitedphenotypic improvement. Our study aimed to measure the PRIMEediting technology efficiency. This technology uses a PRIME editorplasmid (PE2 or PE3) coding for a Moloney murine leukemiavirus reverse transcriptase fused with the Cas9 H840A nickase,and a plasmid coding for a pegRNA containing a primer bindingsites (PBS) and a reverse transcriptase template (RTT). It permitsspecific nucleotide substitutions, deletions or insertions in the genome.We designed different pegRNAs targeting several hDMDexons (9, 20, 35,43, 51, 55, and 61) to introduce a STOP codonby modifying a single nucleotide. HEK293T cells were harvestedfrom DMEM culture media three days after being simultaneouslytransfected with the PE2 and pegRNA. Exons were PCR amplifiedand sequenced using the Sanger method. Results were analysedusing the EditR program to estimate the editing percentage. Weconfirmed that PRIME editing permits the specific C to T andG to T substitutions in the DMD gene with an editing efficiencybetween 6 to 11 % (PE2) and 21% (PE3). Repeated transfections 6days after the first one showed up to 15 % (PE2) edition in exons9 and 35. An additional mutation in PAM sequence (exon 35) improveda PE2 result to 38% for a single transfection. Thus, PRIMEediting permits the specific substitutions in the DMD gene andmight be used to correct point mutations in the DMD gene to leadto dystrophin expression.