A human CAGinSTEM platform for decoding HTT repeat somatic instability links CAG interruption to HD pathology in neurons

Somatic CAG instability in the mutant Huntingtin (HTT) gene is increasingly recognized as a key hallmark of Huntington's disease (HD). Using our novel human CAGinSTEM platform, we manipulated cis genetic elements influencing instability in human HD neurons, monitoring repeat length. Quality-controlled CRISPR-engineered stem cells with increasing CAG lengths and clinical haplotypes were analyzed using third-generation sequencing. Our findings link interruptions in the CAG repeat, especially the loss or duplication of the penultimate CAA of canonical alleles, to significant instability modulation. Notably, four internal CAA interruptions completely abolish CAG instability, reversing HD phenotypes such as altered striatal fate acquisition and nuclear disorganization supported by partial PRC2 loss of function. This platform highlights the role of cis modifiers, emphasizing the direct influence of HTT DNA repeat composition on CAG instability and providing a robust framework for modelling HTT repeat instability in vitro.