Combinatorial and Inducible CRISPRa/i Achieves Deterministic hiPSC Forward Programming and Iterative Refinement via Single-Cell Genomics

Synthetic gene-regulation logic is established in immortalized cell lines but remains largely aspirational in human induced pluripotent stem cells (hiPSCs) and derivatives. This gap constrains both mechanistic discovery and translational engineering in physiologically relevant models. We developed Combinatorial Inducible CRISPR in iPSCs (CIRI), an isogenic, safe-harbor–engineered platform in which tetracycline-responsive single guide RNAs (sgRNAs) carry modular RNA aptamers that recruit RNA-binding proteins and effector domains. CIRI enables multimodal regulation from a single catalytically inactive Cas9 (dCas9), exemplified by orthogonal CRISPR activation and interference (CRISPRa/i). After optimizing sgRNA–aptamer architectures, we achieved robust CRISPRa and CRISPRi in hiPSCs and hiPSC-derived cardiac organoids. CIRI rapidly and deterministically forward programs hiPSCs into skeletal myocytes by activating MYOD1 while repressing NANOG, POU5F1/OCT4, and SOX2. Combinatorial pooled dual-guide single-cell RNA sequencing screens nominate ID3 as a roadblock and KDM6B and SMARCD3 as promoters of myogenic identity. CIRI unlocks programmable synthetic biology in human stem-cell models.