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coli. Despite our efforts to optimize inducer usage, guide RNA (gRNA) architecture and combination, and target gene expression level, the highest gene repression efficiency was 30-50% at the protein level and ∼70% at the mRNA level. The moderate RNA knockdown is possibly caused by the collateral cleavage activity toward bystander RNAs, which acts as a mechanism of type IV-D immunity and perturbs microbial metabolism. Further studies on cellular response to CRISPR/Cas13d and improvement in RNA knockdown efficiency are required prior to practical application of this system in microbes.In Trichoderma reesei, carbon catabolite repression (CCR) significantly downregulates the transcription of cellulolytic enzymes, which is usually mediated by the zinc finger protein Cre1. It was found that there is a conserved region at the C-terminus of Cre1/CreA in several cellulase-producing fungi that contains up to three continuous S/T phosphorylation sites. Here, S387, S388, T389, and T390 at the C-terminus of Cre1 in T. reesei were mutated to valine for mimicking an unphosphorylated state, thereby generating the transformants Tr_Cre1S387V, Tr_Cre1S388V, Tr_Cre1T389V, and Tr_Cre1T390V, respectively. Transcription of cel7a in Tr_ Cre1S388V was markedly higher than that of the parent strain when grown in glucose-containing media. Under these conditions, both filter paperase (FPase) and p-nitrophenyl-β-D-cellobioside (pNPCase) activities, as well as soluble proteins from Tr_Cre1S388V were significantly increased by up to 2- to 3-fold compared with that of other transformants and the parent strain. The results suggested that S388 is critical site of phosphorylation for triggering CCR at the terminus of Cre1. To our knowledge, this is the first report demonstrating an improvement of cellulase production in T. reesei under CCR by mimicking dephosphorylation at the C-terminus of Cre1. Taken together, we developed a precision engineering strategy based on the modification of phosphorylation sites of Cre1 transcription factor to enhance the production of cellulase in T. reesei under CCR.Pectate lyases play an essential role in textiles, animal feed, and oil extraction industries. Pichia pastoris can be an ideal platform for pectate lyases production, and BspPel (a thermo-alkaline pectate lyase from Bacillus sp. RN1) was overexpressed by combined strategies, reaching 1859 U/mL in a 50 L fermentator. It displayed the highest activity at 80°C, and maintained more than 60% of the activity between 30 and 70°C for 1 h. It showed an optimal pH of 10.0, and exhibited remarkable stability over a wider pH range (3.0-11.0), retaining more than 80.0% of enzyme activity for 4 h. The K m and k cat of BspPel on PGA (polygalacturonic acid) was 2.19 g L-1 and 116.1 s-1, respectively. The activity was significantly enhanced by Ca2+, Mn2+, and Cu2+, and a slight increase was observed with the addition of Ba2+ and Mg2+. Scanning electron microscope was used to show the degumming efficiency of BspPel on ramie fibers. The loss weight was 9.2% when treated with crude enzyme supernatant and 20.8% when treated with the enzyme-chemical method, which was higher than the 14.2% weight loss in the positive control treated with 0.5% (w/v) NaOH alone. In conclusion, BspPel could be a good candidate for the ramie degumming industry.Developing non-viral gene therapy vectors that both protect and functionally deliver nucleic acid cargoes will be vital if gene augmentation and editing strategies are to be effectively combined with advanced regenerative medicine approaches. PP242 Currently such methodologies utilize high concentrations of recombinant growth factors, which result in toxicity and off-target effects. Herein we demonstrate the use of modified cell penetrating peptides (CPPs), termed Glycosaminoglycan (GAG)-binding Enhanced Transduction (GET) peptides with plasmid DNA (pDNA) encapsulated poly (lactic-co-glycolic acid) PLGA nanoparticles (pDNA-encapsulated PLGA NPs). In order to encapsulate the pDNA, it was first condensed with a cationic low molecular weight Poly L-Lysine (PLL) into 30-60 nm NPs followed by encapsulation in PLGA NPs by double emulsion; yielding encapsulation efficiencies (EE) of ∼30%. PLGA NPs complexed with GET peptides show enhanced intracellular delivery (up to sevenfold) and transfection efficiencies (up to five orders of magnitude). Moreover, the pDNA cargo has enhanced protection from nucleases (such as DNase I) promoting their translatability. As an example, we show these NPs efficiently deliver pBMP2 which can promote osteogenic differentiation in vitro. Gene delivery to human Mesenchymal Stromal Cells (hMSCs) inducing their osteogenic programming was confirmed by Alizarin red calcium staining and bone lineage specific gene expression (Q RT-PCR). By combining simplistic and FDA-approved PLGA polymer nanotechnology with the GET delivery system, therapeutic non-viral vectors could have significant impact in future cellular therapy and regenerative medicine applications.Isotropic inverse opal structures have been extensively studied for the ability to manipulate cell behaviors such as attachment, migration, and spheroid formation. However, their use in regulate the behaviors of neural stem cells has not been fully explored, besides, the isotropic inverse opal structures usually lack the ability to induce the oriented cell growth which is fundamental in neural regeneration based on neural stem cell therapy. In this paper, the anisotropic inverse opal substrates were obtained by mechanically stretching the poly (vinylidene fluoride) (PVDF) inverse opal films. The anisotropic inverse opal substrates possessed good biocompatibility, optical properties and anisotropy, provided well guidance for the formation of neural spheroids, the alignment of neural stem cells, the differentiation of neural stem cells, the oriented growth of derived neurons and the dendritic complexity of the newborn neurons. Thus, we conclude that the anisotropic inverse opal substrates possess great potential in neural regeneration applications.Cartilage defects pose a great threat to the health of the aging population. Cartilage has limited self-regeneration ability because it lacks blood vessels, nerves and lymph. To achieve efficient cartilage regeneration, supramolecular hydrogels are used in medical applications and tissue engineering as they are tunable and reversible in nature. Moreover, they possess supramolecular interactions which allow the incorporation of cells. These hydrogels present great potential for tissue engineering-based therapies. This review presents advances in the development of stem cell-laden supramolecular hydrogels. We discuss new possibilities for stem cell therapy and their uses in cartilage tissue engineering. Gray areas and future perspectives are discussed.