The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR–FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering.

Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing vessels, is crucial for maintaining tissue homeostasis and immune function. Despite recent advances in understanding the molecular mechanisms regulating lymphangiogenesis, most in vitro studies rely on traditional two-dimensional (2D) cell cultures, with limited replication of the complex microenvironment that governs lymphangiogenesis in vivo. Here, we present a three-dimensional (3D) lymphangiogenesis model using gelatin hydrogels modified with click-chemistry motifs (tetrazine and norbornene, GelTN), providing a biomimetic and mechanically tunable extracellular matrix (ECM) for lymphatic endothelial cells. By encapsulating human dermal lymphatic endothelial cells (HDLEC) spheroids in GelTN, we established a robust and reliable in vitro sprouting assay (<48 h duration) to investigate the effects of GelTN stiffness on lymphangiogenesis. HDLEC encapsulated in low GelTN concentrations exhibited enhanced sprouting in response to vascular endothelial growth factor (VEGF)-C stimulation, compared to HDLEC encapsulated in higher GelTN concentrations. We also provide evidence for the involvement of β3 integrin in lymphangiogenesis. The reduced sprout length upon β3 integrin inhibition further decreased with combined inhibition of α5β1, suggesting a synergistic interaction of the integrin subunits in controlling HDLEC-ECM mechanotransduction. GelTN hydrogels were also evaluated for their translational potential, demonstrating sustained release of VEGF-C in vitro and supporting cellular infiltration and neo-vessel formation following subcutaneous injection in an in vivo mouse model. Overall, these findings highlight the versatility of GelTN hydrogels as a platform for studying lymphangiogenesis and their potential use for therapeutic applications that require controlled growth factor delivery in tissue engineering and regenerative medicine.

Dysregulated lipid metabolism is associated with cardiovascular disease, obesity and type 2 diabetes. In the current report, we explore the functional interactions between two important regulators of lipid metabolism, sterol regulatory element-binding protein 1 and 2 (SREBP1/2), and PRDI-BF1 and RIZ homology domain containing 16 (PRDM16). The SREBP family of transcription factors regulate cholesterol and fatty acid synthesis and metabolism, primarily in liver but also in white adipose tissue. PRDM16 is a major regulator of brown adipose tissue (BAT) biogenesis and function as well as an inhibitor of white adipogenesis. We find that PRDM16 interacts with the nuclear forms of SREBP1/2 and inhibits their transcriptional activities. Consequently, inactivation of PRDM16 enhances the expression of well-established SREBP target genes involved in fatty acid and cholesterol synthesis/metabolism. Importantly, PRDM16 inactivation increases the expression of LDL receptor mRNA (1.6-fold) and protein (1.7-fold) and augments the cellular uptake of LDL particles (2.3-fold). Supporting these findings, PRDM16-deficient cells accumulate more neutral lipids in a SREBP1/2-dependent manner. Inactivation of PRDM16 in white and brown preadipocyte cell lines and human adipose-derived stem cells enhances the expression of SREBP target genes. In addition, the expression of adipogenic markers was increased in mature white adipocytes generated from PRDM16-knockdown preadipocytes (1.8- to 3.9-fold). Thus, our study identifies PRDM16 as a novel inhibitor of SREBP-dependent lipid metabolism with implications for adipose biology and metabolic disease.

Transcription factor networks are crucial for the regulation of endothelial cell gene expression during vascular development and homeostasis. A recent analysis of 269 rare diseases in 77,539 individuals revealed an association between loss-of-function variants in ERG, encoding an ETS transcription factor, with primary lymphoedema. However, the pathogenicity of such variants and possible mechanisms of ERG-associated lymphatic vessel dysfunction remains to be established. Here, we have further identified and characterised lymphoedema-associated ERG genetic variants, revealing pathogenic mechanisms ranging from differential ERG subcellular localisation to altered DNA-binding and impaired transactivation. We confirm a role for ERG in regulating lymphangiogenesis using in vitro assays and a lymphatic endothelium-specific Erg deletion mouse model. Furthermore, we characterise the transcriptional and epigenomic landscape of human dermal lymphatic endothelial cells, identifying a unique role for ERG in regulating lymphatic gene programs, including the establishment of cooperative TF networks with PROX1 and GATA2. Our studies identify ERG as a master regulator of lymphatic endothelial cell transcriptional networks and uncover the mechanisms that underpin a novel causative gene for primary lymphoedema.

Dysregulated lipid metabolism is associated with cardiovascular disease, obesity and type 2 diabetes. In the current report we explore the functional interactions between two im-portant regulators of lipid metabolism, sterol regulatory element-binding protein 1 and 2 (SREBP1/2) and PRDI-BF1 and RIZ homology domain containing 16 (PRDM16). The SREBP family of transcription factors regulate cholesterol and fatty acid synthesis and metabolism, primarily in liver but also in white adipose tissue. PRDM16 is a major regulator of brown adipose tissue (BAT) biogenesis and function, and an inhibitor of white adipogenesis. We find that PRDM16 interacts with the nuclear forms of SREBP1/2 and inhibits their transcriptional activities. Consequently, inactivation of PRDM16 enhances the expression of well-established SREBP target genes involved in fatty acid and cho-lesterol synthesis/metabolism. Importantly, PRDM16 inactivation increases the expression of LDL receptor mRNA and protein and augments the cellular uptake of LDL particles. Supporting these findings, PRDM16-deficient cells accumulate more neutral lipids in a SREBP1/2-dependent manner. Inactivation of PRDM16 in white and brown preadipocyte cell lines and human adipose-derived stem cells enhances the expression of SREBP target genes. In addition, the expression of adipogenic markers was increased in mature white adipocytes generated from PRDM16-knockdown preadipocytes. Thus, our study identifies PRDM16 as a novel inhibitor of SREBP-dependent lipid metabolism with implications for adipose biology and metabolic disease.