Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.

The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) regulates EGF‐receptor and TNFα signaling, thereby not only protecting the skin and intestinal barrier, but also contributing to autoimmunity. ADAM17 can be rapidly activated by many stimuli through its transmembrane domain (TMD), with the seven membrane‐spanning inactive Rhomboids (iRhom) 1 and 2 implicated as candidate regulatory partners. However, several alternative models of ADAM17 regulation exist that do not involve the iRhoms, such as regulation through disulfide bond exchange or through interaction with charged phospholipids. Here, we report that a non‐activatable mutant of ADAM17 with the TMD of betacellulin (BTC) can be rescued by restoring residues from the ADAM17 TMD, but only in Adam17−/− cells, which contain iRhoms, not in iRhom1/2−/− cells. We also provide the first evidence that the extracellular juxtamembrane domains (JMDs) of ADAM17 and iRhom2 regulate the stimulation and substrate selectivity of ADAM17. Interestingly, a point mutation in the ADAM17 JMD identified in a patient with Tetralogy of Fallot, a serious heart valve defect, affects the substrate selectivity of ADAM17 toward Heparin‐binding epidermal growth factor like growth factor (HB‐EGF), a crucial regulator of heart valve development in mice. These findings provide new insights into the regulation of ADAM17 through an essential interaction with the TMD1 and JMD1 of iRhom2.

Aims The precise cellular identity and molecular features of non-myocytes (nonCM) in a mammalian heart at a single cell level remain elusive. Depiction of epigenetic landscape with transcriptomic signatures using the latest single-cell multi-omics has the potential to unravel the molecular programs underlying the cellular diversity of cardiac non-myocytes. Here, we characterized the molecular and cellular features of cardiac nonCM populations in the adult murine heart at the single cell level. Methods and Results Through single-cell dual omics analysis, we mapped the epigenetic landscapes, characterized the transcriptomic profiles and delineated the molecular signatures of cardiac nonCMs in the adult murine heart. Distinct cis-regulatory elements and trans-acting factors for the individual major nonCM cell types (endothelial cells, fibroblast, pericytes and immune cells) were identified. In particular, unbiased sub-clustering and functional annotation of cardiac fibroblasts (FB) revealed extensive FB heterogeneity and identified FB subtypes with functional states related to cellular response to stimuli, cytoskeleton organization and immune regulation, respectively. We further explored the function of marker genes Hsd11b1 and Gfpt2 that label major FB sub-populations and determined the distribution of Hsd11b1+ and Gfp2+ FBs in murine healthy and diseased hearts. Conclusions In summary, we characterized the nonCM cellular identity at the transcriptome and epigenome levels using single-cell omics approaches and discovered previously unrecognized cardiac fibroblast subpopulations with unique functional states. Translational Perspective Our research identified discrete cell types of nonCM in the heart and differentially expressed genes with regulatory factors. Unveiling the heterogeneity of nonCMs and molecular signatures of each cell type or subtypes allows for study, precise capture and manipulation of specific cell type(s) in heart and will provide insights into the development of therapeutics for cardiovascular diseases.

Direct reprogramming of fibroblasts to alternative cell fates by forced expression of transcription factors offers a platform to explore fundamental molecular events governing cell fate identity. The discovery and study of induced cardiomyocytes (iCMs) not only provides alternative therapeutic strategies for heart disease but also sheds lights on basic biology underlying CM fate determination. The iCM field has primarily focused on early transcriptome and epigenome repatterning, whereas little is known about how reprogramming iCMs remodel, erase, and exit the initial fibroblast lineage to acquire final cell identity. Here, we show that autophagy-related 5 (Atg5)–dependent autophagy, an evolutionarily conserved self-digestion process, was induced and required for iCM reprogramming. Unexpectedly, the autophagic factor Beclin1 (Becn1) was found to suppress iCM induction in an autophagy-independent manner. Depletion of Becn1 resulted in improved iCM induction from both murine and human fibroblasts. In a mouse genetic model, Becn1 haploinsufficiency further enhanced reprogramming factor–mediated heart function recovery and scar size reduction after myocardial infarction. Mechanistically, loss of Becn1 up-regulated Lef1 and down-regulated Wnt inhibitors, leading to activation of the canonical Wnt/β-catenin signaling pathway. In addition, Becn1 physically interacts with other classical class III phosphatidylinositol 3-kinase (PI3K III) complex components, the knockdown of which phenocopied Becn1 depletion in cardiac reprogramming. Collectively, our study revealed an inductive role of Atg5-dependent autophagy as well as a previously unrecognized autophagy-independent inhibitory function of Becn1 in iCM reprogramming.

Mutton fat has a similar fatty acid profile with Cocoa Butter (CB), except that its degree of unsaturation of Triacylglycerol (TAG) at the Sn-2 position is considerably lower than CB and maybe increased by Sn-2 specific lipase to produce Cocoa Butter Equivalent (CBE), a healthy functional lipid. However, there is no commercially available Sn-2 specific lipase that can be used to convert mutton fat to CBE by improving its Unsaturated Fatty Acids (UFA) at Sn-2 position. Similar to plant, yeast fat contains higher UFAs at the Sn-2 position than animal fat. In this study, we investigated the conversion of mutton fat to CBE by fermentation of oleaginous yeast Yarrowia lipolytica which acts as a "Sn-2 specific lipase". The yeast was able to grow on mutton fat as the sole carbon source yielding a dry cell weight of 14.11 g L −1 and 33.1% lipid content after 3 days of cultivation. At optimal fermentation conditions, the degree of unsaturation of TAGs at the Sn-2 position increased from 61.5 (mutton fat) to 89.3% (cellar lipid, 72 h) while the amount of Saturated Fatty Acids (SFA) of the Total Fatty Acids (TFA) was decreased from 58.9 to 34.5%. In addition, the presence of methyl stearate as the cosubstrate in the medium improved the ratio of SFAs/TFAs. It was found that fatty acid profile of the yeast fat with 24.60% palmitic acid, 31.34% stearic acid, 34.29% oleic acid, 5.57% linoleic acid and degree of unsaturation at Sn-2 position in TAGs (84.66%) resembled that of CB when the yeast was grown on mutton fat/methyl stearate (with a ratio of 60/40) as carbon source. These results suggest that biotransformation or metabolism could be directed by using mixtures of inexpensive animal fats and saturated fatty acid or methyl as co-substrates, to produce functional lipids with predetermined composition, such as CBE.