INNOVATION IN CRISPR GENOMIC EDITING IN HUMAN PLURIPOTENT STEM CELLS

Human synthetic biology aspires to mimic the sophistication of naturally occurring gene regulation. Emerging methods based on programmable genome modifiers are optimized for immortalized cell lines, which cannot model the breadth of human pathophysiology. Implementation of these approaches in human induced pluripotent stem cells (hiPSCs) and their derivatives is highly desirable, but several challenges remain. To address this technological gap, we developed CIRI (Combinatorial Inducible cRispr in Ipscs), a multimodal CRISPR/dCas9-based method to conditionally manipulate complex genetic circuits in hPSCs and their differentiated derivatives. CIRI leverages tetracycline-inducible single guide RNAs (sgRNAs) engineered to include modular RNA aptamers able to recruit effectors at target genomic sites. This bypasses the need for big dCas9 fusion proteins. Moreover, by combining different aptamers, RNA binding proteins, and effectors, CIRI allows the simultaneous execution of distinct regulatory activities at multiple loci; i.e., activating a set of genes (CRISPRa) while others are being repressed (CRISPRi). sgRNAs also contain distinct capture sequences that facilitate their identification in the context of pooled screens via single-cell RNA sequencing (scRNA-seq). Finally, all genetic instructions are inserted in genomic safe harbors to ensure stable expression. We first tested variations in sgRNA structures and effectors, and identified strategies that allow strong CRISPRa and CRISPRi in hiPSCs. We then deployed CIRI to forward program hPSCs into skeletal myocytes through the combined repression of NANOG, OCT4, and SOX2 and the activation of MYOD1. Lastly, we developed methods for single- and dual-guide combinatorial scRNA-seq screens based on CIRI, and conducted proof-of-principle experiments to identify roadblocks and promoters of muscle maturation. We anticipate that CIRI will facilitate precise and scalable gene control for diverse applications in functional genomics and synthetic biology