Protocol for generating endogenous degron tags in essential transcription factors in human iPSCs via CRISPR-Cas9

Precise genome engineering in human pluripotent stem cells remains inefficient, limiting endogenous fluorescent tagging needed to study phase-separated membraneless nuclear compartments. Here, we present a protocol for generating precise knockin alleles of essential genes in human induced pluripotent stem cells (iPSCs) via CRISPR-Cas9 editing. We describe steps for combining transient p53 inhibition, optimized transfection conditions, and fluorescence-activated cell sorting. This protocol enables recovery of viable edited clones that would otherwise be lost due to editing-induced stress or essential gene perturbation. For complete details on the use and execution of this protocol, please refer to Li et al.¹.