Prime editing corrects G6PD deficiency in stem cells

G6PD deficiency is an X-linked enzyme defect that compromises erythrocyte redox defence and predisposes to acute haemolysis under oxidative stress. G6PD sits on the X chromosome and supplies NADPH via the pentose phosphate pathway; deficiency leaves red cells vulnerable to reactive oxygen species, with haemolytic crises triggered by infections, certain medicines and particular foods.

There is no definitive therapy, and management remains supportive. Among >200 variants, G6PD Viangchan (c.871G>A, Lys→Met in exon 9) predominates in several South-East Asian populations and is associated with severe enzymatic impairment with <10% normal activity.

In this study, the Thai authors modelled and corrected Viangchan using CRISPR prime editing. First, a HEK293T model bearing G6PD Viangchan (c.871G>A) was created. Then, they optimised plasmid PE3 by varying epegRNA primer-binding site (PBS) and reverse transcription template (RTT) lengths as well as the position of the secondary nick.

In addition, PE5 (incorporating an engineered dominant-negative MLH1 mutant, MLH1dn) was benchmarked against PE3 at the same locus. Editing and by-products were quantified by Sanger/TIDER and targeted NGS, and predicted off-target sites for the epegRNA and the +64 nicking guide were interrogated by deep sequencing.

The optimised PE3 design – epegRNA with a 14-nt primer-binding site and 19-nt RTT plus a secondary nicking sgRNA at +64 – achieved ~23–25% intended edits in HEK293T, whereas PE5 reduced yields. In G6PD-deficient CHULAi001-A iPSCs, bulk correction was 3.0% by NGS and increased to ~5.0% after GFP sorting, with by-products near baseline and no excess mutations at predicted off-target loci by NGS/TIDE analyses.

A single silent change adjacent to the pathogenic site unexpectedly lowered efficiency about fourfold, underscoring locus-specific constraints in epegRNA design. Functional rescue in erythroid derivatives was not assessed owing to current limitations in iPSC erythroid maturation protocols.

The research was led by Natee Jearawiriyapaisarn and Kamonlak Leecharoenkiat at Mahidol University and Chulalongkorn University, respectively, in Thailand. It was published in Scientific Reports on 18 August 2025.