SUMMARY Pluripotent stem cell (PSC)-derived organoids have emerged as novel multicellular models of human tissue development but display immature phenotypes, aberrant tissue fates, and a limited subset of cells. Here, we demonstrate that integrated analysis and engineering of gene regulatory networks (GRNs) in PSC-derived multilineage human liver organoids direct maturation and vascular morphogenesis in vitro . Overexpression of PROX1 and ATF5 , combined with targeted CRISPR-based transcriptional activation of endogenous CYP3A4 , reprograms tissue GRNs and improves native liver functions, such as FXR signaling, CYP3A4 enzymatic activity, and stromal cell reactivity. The engineered tissues possess superior liver identity when compared with other PSC-derived liver organoids and show the presence of hepatocyte, biliary, endothelial, and stellate-like cell populations in single-cell RNA-seq analysis. Finally, they show hepatic functions when studied in vivo . Collectively, our approach provides an experimental framework to direct organogenesis in vitro by systematically probing molecular pathways and transcriptional networks that promote tissue development.

Transient modulation of genes involved in immunity, without exerting a permanent change in the DNA code, can be an effective strategy to modulate the course of many inflammatory conditions. CRISPR-Cas9 technology represents a promising platform for achieving this goal. Truncation of guide RNA (gRNA) from 5’ end, enables the application of a nuclease competent Cas9 protein for transcriptional modulation of genes, allowing multi-functionality of CRISPR. Here, we introduce an enhanced CRISPR-based transcriptional repressor to reprogram immune homeostasis in vivo . In this repressor system, two transcriptional repressors heterochromatin protein 1 (HP1a) and Krüppel associated box (KRAB) are fused to MS2 coat protein and subsequently recruited by gRNA aptamer binding to a nuclease competent CRISPR complex containing truncated gRNAs. With the enhanced repressor, we demonstrate transcriptional repression of the Myeloid differentiation primary response 88 ( Myd88 ) gene in vitro and in vivo. We demonstrate that this strategy can efficiently downregulate Myd88 expression in lung, blood and bone marrow of Cas9 transgenic mice, which receive systemic injection of adeno-associated virus- (AAV)2/1 carrying truncated gRNAs targeting Myd88 and MS2-Hp1aKRAB cassette. This downregulation is accompanied by changes in downstream signaling elements such as TNF-α and ICAM-1. Myd88 repression leads to decrease in immunoglobulin G (IgG) production against AAV2/1 and AAV2/9 and the strategy modulates IgG response against AAV cargos. It improves the efficiency of a subsequent AAV9/CRISPR treatment for repression of Proprotein convertase subtilisin/kexin type 9 ( PCSK9 ), a gene when repressed can lower blood cholesterol levels. We also demonstrate that CRISPR-mediated Myd88 repression can act as a prophylactic measure against septicemia in both Cas9 transgenic and C57BL/6J mice. When delivered by nanoparticles, this repressor can serve as a therapeutic modality to influence the course of septicemia. Collectively, we report that CRISPR-mediated repression of endogenous Myd88 can effectively modulate host immune response against AAV-mediated gene therapy and influence the course of septicemia. The ability to control Myd88 transcript levels using a CRISPR-based synthetic repressor can be an effective strategy for AAV-based CRISPR therapies, as this pathway serves as a key node in induction of humoral immunity against AAV serotypes.

Graphical AbstractHIGHLIGHTS  In vitro tissue maturation via genetically encoded molecular programs  Computational analysis to identify maturation transcription factors in liver organoids  Promoting vascularization of organoids via genetically encoded molecular programs  Single cell analysis of parenchymal and non-parenchymal cells  Modeling of native liver functions and in vivo therapeutic potential SUMMARY Pluripotent stem cell (PSC)-derived organoids are emerging as novel human-based microphysiological models but display immature phenotypes with limited subsets of endothelial or stromal cells. Here we demonstrate that in vitro manipulation of gene regulatory networks (GRNs) in PSC-derived liver organoids selected either through computational analysis or targeted tissue design can advance tissue maturation in vitro. Through an unbiased comparison with the genetic signature of mature livers, we identify downregulated GRNs in fetal liver organoids compared to adult livers. We demonstrate that overexpression of PROX1 and ATF5, together with targeted CRISPR-based transcriptional activation of endogenous CYP3A4, drives maturation in vitro. Single cell analyses reveal hepatobiliary-, endothelial-, and stellate-like cell populations. The engineered organoids demonstrate enhanced vasculogenesis, capture native liver characteristics (e.g. FXR signaling, CYP3A4 activity), and exhibit therapeutic potential in mice. Collectively, our approach provides a genetically guided framework for engineering developmentally advanced multilineage tissues from hiPSCs.