Preprint details cytosine base editing workflow for validated multiplex-knockout hiPSC lines

A preprint on bioRxiv presents a systematic workflow for producing multiplex-knockout (KO) human induced pluripotent stem cell (hiPSC) lines using cytosine base editing — a form of CRISPR-based genome editing that converts cytosine bases to uracil (read as thymine) at a target site, without making double-strand DNA breaks. The authors evaluated six cytosine base editor variants and selected evoBE4max as the most efficient, then combined it with single guide RNA-directed introduction of premature stop codons and splice-site disruptions, followed by fluorescence-based cell enrichment to select edited cells.

The workflow addresses a significant bottleneck in human disease modelling: dissecting the molecular mechanisms of polygenic diseases requires cell models with multiple targeted genetic modifications in a defined genetic background, but generating and rigorously validating such multi-KO lines has been technically challenging. The preprint reports that the method achieves a median on-target editing efficiency sufficient for practical research use, and provides a quality-control framework covering off-target assessment and genomic integrity checks.

hiPSC-based models are widely used in neurodevelopmental and metabolic disease research, and multiplex KO capability is particularly relevant for functional follow-up of GWAS and rare-variant findings. The preprint has not yet been peer reviewed.