Compact engineered human mechanosensitive transactivation modules enable potent and versatile synthetic transcriptional control.

Engineered transactivation domains (TADs) combined with programmable DNA binding platforms have revolutionized synthetic transcriptional control. Despite recent progress in programmable CRISPR-Cas-based transactivation (CRISPRa) technologies, the TADs used in these systems often contain poorly tolerated elements and/or are prohibitively large for many applications. Here, we defined and optimized minimal TADs built from human mechanosensitive transcription factors. We used these components to construct potent and compact multipartite transactivation modules (MSN, NMS and eN3x9) and to build the CRISPR-dCas9 recruited enhanced activation module (CRISPR-DREAM) platform. We found that CRISPR-DREAM was specific and robust across mammalian cell types, and efficiently stimulated transcription from diverse regulatory loci. We also showed that MSN and NMS were portable across Type I, II and V CRISPR systems, transcription activator-like effectors and zinc finger proteins. Further, as proofs of concept, we used dCas9-NMS to efficiently reprogram human fibroblasts into induced pluripotent stem cells and demonstrated that mechanosensitive transcription factor TADs are efficacious and well tolerated in therapeutically important primary human cell types. Finally, we leveraged the compact and potent features of these engineered TADs to build dual and all-in-one CRISPRa AAV systems. Altogether, these compact human TADs, fusion modules and delivery architectures should be valuable for synthetic transcriptional control in biomedical applications.

Lipid Deposition Profiles Influence Foreign Body Responses.

Fibrosis remains a significant cause of failure in implanted biomedical devices and early absorption of proteins on implant surfaces has been shown to be a key instigating factor. However, lipids can also regulate immune activity and their presence may also contribute to biomaterial-induced foreign body responses (FBR) and fibrosis. Here it is demonstrated that the surface presentation of lipids on implant affects FBR by influencing reactions of immune cells to materials as well as their resultant inflammatory/suppressive polarization. Time-of-flight secondary ion mass spectroscopy (ToF-SIMS) is employed to characterize lipid deposition on implants that are surface-modified chemically with immunomodulatory small molecules. Multiple immunosuppressive phospholipids (phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin) are all found to deposit preferentially on implants with anti-FBR surface modifications in mice. Significantly, a set of 11 fatty acids is enriched on unmodified implanted devices that failed in both mice and humans, highlighting relevance across species. Phospholipid deposition is also found to upregulate the transcription of anti-inflammatory genes in murine macrophages, while fatty acid deposition stimulated the expression of pro-inflammatory genes. These results provide further insights into how to improve the design of biomaterials and medical devices to mitigate biomaterial material-induced FBR and fibrosis.

Systematic comparison of CRISPR-based transcriptional activators uncovers gene-regulatory features of enhancer-promoter interactions.

Nuclease-inactivated CRISPR/Cas-based (dCas-based) systems have emerged as powerful technologies to synthetically reshape the human epigenome and gene expression. Despite the increasing adoption of these platforms, their relative potencies and mechanistic differences are incompletely characterized, particularly at human enhancer-promoter pairs. Here, we systematically compared the most widely adopted dCas9-based transcriptional activators, as well as an activator consisting of dCas9 fused to the catalytic core of the human CBP protein, at human enhancer-promoter pairs. We find that these platforms display variable relative expression levels in different human cell types and that their transactivation efficacies vary based upon the effector domain, effector recruitment architecture, targeted locus and cell type. We also show that each dCas9-based activator can induce the production of enhancer RNAs (eRNAs) and that this eRNA induction is positively correlated with downstream mRNA expression from a cognate promoter. Additionally, we use dCas9-based activators to demonstrate that an intrinsic transcriptional and epigenetic reciprocity can exist between human enhancers and promoters and that enhancer-mediated tracking and engagement of a downstream promoter can be synthetically driven by targeting dCas9-based transcriptional activators to an enhancer. Collectively, our study provides new insights into the enhancer-mediated control of human gene expression and the use of dCas9-based activators.

CRISPR/Cas-Based Epigenome Editing: Advances, Applications, and Clinical Utility.

The epigenome dynamically regulates gene expression and guides cellular differentiation throughout the lifespan of eukaryotic organisms. Recent advances in clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based epigenome editing technologies have enabled researchers to site-specifically program epigenetic modifications to endogenous DNA and histones and to manipulate the architecture of native chromatin. As a result, epigenome editing has helped to uncover the causal relationships between epigenetic marks and gene expression. As epigenome editing tools have continued to develop, researchers have applied them in new ways to explore the function of the epigenome in human health and disease. In this review, we discuss the recent technical improvements in CRISPR/Cas-based epigenome editing that have advanced clinical research and examine how these technologies could be improved for greater future utility.

Programmable human histone phosphorylation and gene activation using a CRISPR/Cas9-based chromatin kinase.

Histone phosphorylation is a ubiquitous post-translational modification that allows eukaryotic cells to rapidly respond to environmental stimuli. Despite correlative evidence linking histone phosphorylation to changes in gene expression, establishing the causal role of this key epigenomic modification at diverse loci within native chromatin has been hampered by a lack of technologies enabling robust, locus-specific deposition of endogenous histone phosphorylation. To address this technological gap, here we build a programmable chromatin kinase, called dCas9-dMSK1, by directly fusing nuclease-null CRISPR/Cas9 to a hyperactive, truncated variant of the human MSK1 histone kinase. Targeting dCas9-dMSK1 to human promoters results in increased target histone phosphorylation and gene activation and demonstrates that hyperphosphorylation of histone H3 serine 28 (H3S28ph) in particular plays a causal role in the transactivation of human promoters. In addition, we uncover mediators of resistance to the BRAF V600E inhibitor PLX-4720 in human melanoma cells using genome-scale screening with dCas9-dMSK1. Collectively, our findings enable a facile way to reshape human chromatin using CRISPR/Cas9-based epigenome editing and further define the causal link between histone phosphorylation and human gene activation.

THz imaging system for in vivo human cornea.

Terahertz (THz) imaging of corneal tissue water content (CTWC) is a proposed method for early, accurate detection and study of corneal diseases. Despite promising results from ex vivo and in vivo cornea studies, interpretation of the reflectivity data is confounded by the contact between corneal tissue and rigid dielectric window used to flatten the imaging field. This work develops a novel imaging system and image reconstruction methods specifically for nearly spherical targets such as human cornea. A prototype system was constructed using a 650 GHz multiplier source and Schottky diode detector. Resolution and imaging field strength measurement from characterization targets correlate well with those predicted by the quasioptical theory and physical optics analysis. Imaging experiments with corneal phantoms and ex vivo corneas demonstrate the hydration sensitivity of the imaging system and reliable measurement of CTWC. We present successful acquisition of non-contact THz images of in vivo human cornea, and discuss strategies for optimizing the imaging system design for clinical use.

Optical System Design for Noncontact, Normal Incidence, THz Imaging of in vivo Human Cornea.

Reflection mode Terahertz (THz) imaging of corneal tissue water content (CTWC) is a proposed method for early, accurate detection and study of corneal diseases. Despite promising results from ex vivo and in vivo cornea studies, interpretation of the reflectivity data is confounded by the contact between corneal tissue and dielectric windows used to flatten the imaging field. Herein, we present an optical design for non-contact THz imaging of cornea. A beam scanning methodology performs angular, normal incidence sweeps of a focused beam over the corneal surface while keeping the source, detector, and patient stationary. A quasioptical analysis method is developed to analyze the theoretical resolution and imaging field intensity profile. These results are compared to the electric field distribution computed with a physical optics analysis code. Imaging experiments validate the optical theories behind the design and suggest that quasioptical methods are sufficient for designing of THz corneal imaging systems. Successful imaging operations support the feasibility of non-contact in vivo imaging. We believe that this optical system design will enable the first, clinically relevant, in vivo exploration of CTWC using THz technology.