CIRI: a discovery platform for genetic programming
We developed CIRI (Combinatorial Inducible CRISPR in iPSCs), a multimodal system that enables precise, reversible, and combinatorial gene regulation in human pluripotent stem cells and their derivatives. CIRI integrates inducible CRISPRa/i modules with single-cell multi-omics readouts, allowing simultaneous activation and repression of distinct gene sets across differentiation trajectories. Unlike conventional dCas9 fusion systems, CIRI employs a modular RNA design that is compact and compatible with genome-safe integration sites. The platform supports both pooled discovery screens and arrayed validation studies, enabling systematic exploration of how combinations of genome architects influence lineage choice.
In proof-of-concept experiments, CIRI successfully programmed pluripotent stem cells into skeletal myocytes by coordinating activation of myogenic regulators and repression of pluripotency factors. These studies demonstrate the potential of CIRI to identify synthetic, yet physiologically faithful, gene circuits based on candidate genome architects for programming cell differentiation.
In proof-of-concept experiments, CIRI successfully programmed pluripotent stem cells into skeletal myocytes by coordinating activation of myogenic regulators and repression of pluripotency factors. These studies demonstrate the potential of CIRI to identify synthetic, yet physiologically faithful, gene circuits based on candidate genome architects for programming cell differentiation.
Overview of CIRI's key features and applications. From Sozza F., PhD thesis & PCT/IB2025/055835
GERALT: a production platform for scalable differentiation
We established GERALT (Genetically Enhanced Renewal and Autonomous Lineage Transdifferentiation) as a production platform for low-cost and streamlined stem-cell expansion and differentiation. GERALT implements a genetically encoded circuit that couples self-renewal with inducible lineage conversion, removing the dependence on external growth factors and improving scalability. When activated, the system synchronously silences a previously activated pluripotency genome architects while inducing lineage-specific genome architects, producing highly homogeneous differentiation within days under chemically defined conditions, demonstrated as proof-of-principle for skeletal muscle.
Together, CIRI and GERALT embody the writing phase of our Genome Architecting framework: one discovers the combinations of genome architects that can control cell fate, the other implements them in a stable, efficient, and scalable format. This approach moves synthetic biology beyond editing individual genes—toward programming genome logic itself to achieve scalble, rapid, and cost-effective differentiation of mature cells.
Together, CIRI and GERALT embody the writing phase of our Genome Architecting framework: one discovers the combinations of genome architects that can control cell fate, the other implements them in a stable, efficient, and scalable format. This approach moves synthetic biology beyond editing individual genes—toward programming genome logic itself to achieve scalble, rapid, and cost-effective differentiation of mature cells.
Overview of GERALT and examples of suspension cultures (right, TTN-mEGFP reporter). From Bottini S., PhD thesis & PCT/IB2025/055837