The CRISPR-Cas9 system has raised hopes for developing personalized gene therapies for complex diseases. Its application for genetic and epigenetic therapies in humans raises concerns over immunogenicity of the bacterially derived Cas9 protein. Here we detect antibodies to Streptococcus pyogenes Cas9 (SpCas9) in at least 5% of 143 healthy individuals. We also report pre-existing human CD8+T cell immunity in the majority of healthy individuals screened. We identify two immunodominant SpCas9 T cell epitopes for HLA-A*02:01 using an enhanced prediction algorithm that incorporates T cell receptor contact residue hydrophobicity and HLA binding and evaluated them by T cell assays using healthy donor PBMCs. In a proof-of-principle study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity. Our study highlights the problem of pre-existing immunity against CRISPR-associated nucleases and offers a potential solution to mitigate the T cell immune response.

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.

Highlights • Liposomal formulation in this research had a better function than Lipofectamine® 2000. • The average particle size had no significant change while the PDI increased after lyophilization. • LacZ expression of the developed cationic liposomes is approximately equal to the Lipofectamine® 2000.

An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence. CRISPR systems open-up widespread applications including genetic disease modeling, functional screens, and synthetic gene regulation. The plausibility of in vivo genetic engineering using CRISPR has garnered significant traction as a next generation in vivo therapeutic. However, there are hurdles that need to be addressed before CRISPR-based strategies are fully implemented. Some key issues center on the controllability of the CRISPR platform, including minimizing genomic-off target effects and maximizing in vivo gene editing efficiency, in vivo cellular delivery, and spatial-temporal regulation. The modifiable components of CRISPR systems: Cas9 protein, gRNA, delivery platform, and the form of CRISPR system delivered (DNA, RNA, or ribonucleoprotein) have recently been engineered independently to design a better genome engineering toolbox. This review focuses on evaluating CRISPR potential as a next generation in vivo gene therapy platform and discusses bioengineering advancements that can address challenges associated with clinical translation of this emerging technology.

The application of Cas9 for genetic and epigenetic therapies in humans raises concerns over immunogenicity of this foreign protein. We report pre-existing human CD8+ T cell immunity to Streptococcus pyogenes Cas9 in the majority of healthy individuals screened. In a proof-ofprinciple study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity.Of the Cas9 orthologs derived from bacterial species, the SpCas9 is the best characterized. S. pyogenes is a ubiquitous pathogen, with an annual incidence of 700 million worldwide 26 , but immunity to SpCas9 in humans has not been reported. Here, we sought to characterize the pre-existing immune response to SpCas9 in healthy individuals and to identify the immunodominant T cell epitopes with the aim of developing SpCas9 proteins that have diminished capacity to invoke human adaptive response.CRISPR application for human therapies will span its use both for gene editing (through DNA double-strand breaks) or epigenetic therapies (without DNA double-strand breaks). In fact, recent reports shed light on CRISPR's ability to activate or repress gene expression in mice [27][28][29] , which opens the door to a variety of new therapeutic applications such as activating silent genes, compensating for disrupted genes, cell fate reprogramming, or silencing disrupted genes, without the concern over permanent change in DNA sequence. However, unlike the use of Cas9 for gene editing, which may only require Cas9 presence in cells for a few hours, current techniques for CRISPR-based epigenetic therapies require longer term expression of Cas9 in vivo, possibly for weeks and months 28,29 , which poses the challenge of combating pre-existing immune response towards Cas9. This challenge will need to be addressed before CRISPR application for human therapies, especially for epigenetic therapies, can be fully implemented. Delivery of CRISPR in vivo by incorporating its expression cassette in adeno-associated virus (AAV), will most likely shape many of the initial clinical trials as AAV-based gene delivery is one of the safest and most prevalent forms of gene therapies in human. AAV will enable longer term expression of Cas9, desirable for epigenetic therapies. Therefore, unlike Cas9 delivery in the form of ribonucleoprotein complexes (which are short term), it is highly likely that CRISPR delivery through AAV and its expression within target cells will engage CD8+ T cell immunity.

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.

The genus Pantoea is a predominant member of host-associated microbiome. We here report on the genomic analysis of Pantoea eucrina strain Russ that was isolated from a trashcan at Oklahoma State University, Stillwater, OK. The draft genome of Pantoea eucrina strain Russ consists of 3,939,877 bp of DNA with 3704 protein-coding genes and 134 RNA genes. This is the first report of a genome sequence of a member of Pantoea eucrina. Genomic analysis revealed metabolic versatility with genes involved in the metabolism and transport of all amino acids as well as glucose, fructose, mannose, xylose, arabinose and galactose, suggesting the organism is a versatile heterotroph. The genome also encodes an extensive secretory machinery including types I, II, III, IV, and Vb secretion systems, and several genes for pili production including the new usher/chaperone system (pfam 05,229). The implications of these systems for opportunistic pathogenesis are discussed.

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.