Enhanced Calvarial Bone Repair Using ASCs Engineered with RNA-Guided Split dCas12a System that Co-Activates Sox 5, Sox6, and Long Non-Coding RNA H19.

Healing of large calvarial bone defects remains challenging. An RNA-guided Split dCas12a system is previously harnessed to activate long non-coding RNA H19 (lncRNA H19, referred to as H19 thereafter) in bone marrow-derived mesenchymal stem cells (BMSCs). H19 activation in BMSCs induces chondrogenic differentiation, switches bone healing pathways, and improves calvarial bone repair. Since adipose-derived stem cells (ASCs) can be harvested more easily in large quantity, here it is aimed to use ASCs as an alternative cell source. However, H19 activation alone using the Split dCas12a system in ASCs failed to elicit evident chondrogenesis. Therefore, split dCas12a activators are designed more to co-activate other chondroinductive transcription factors (Sox5, Sox6, and Sox9) to synergistically potentiate differentiation. It is found that co-activation of H19/Sox5/Sox6 in ASCs elicited more potent chondrogenic differentiation than activation of Sox5/Sox6/Sox9 or H19 alone. Co-activating H19/Sox5/Sox6 in ASCs significantly augmented in vitro cartilage formation and in vivo calvarial bone healing. These data altogether implicated the potentials of the Split dCas12a system to trigger multiplexed gene activation in ASCs for differentiation pathway reprogramming and tissue regeneration.

Split dCas12a activator for lncRNA H19 activation to enhance BMSC differentiation and promote calvarial bone healing.

Healing of large calvarial bone defects in adults is challenging. We previously showed that inducing chondrogenic differentiation of mesenchymal stem cells from bone marrow (BMSC) or adipose tissue (ASC) before implantation can switch the repair pathway and improve calvarial bone healing. Split dCas12a activator is a new CRISPR activation system comprising the amino (N) and carboxyl (C) fragments of dCas12a protein, each being fused with synthetic transcription activators at both termini. The split dCas12a activator was shown to induce programmable gene expression in cell lines. Here we exploited the split dCas12a activator to activate the expression of chondroinductive long non-coding RNA H19. We showed that co-expression of the split N- and C-fragments resulted in spontaneous dimerization, which elicited stronger activation of H19 than full-length dCas12a activator in rat BMSC and ASC. We further packaged the entire split dCas12a activator system (13.2 kb) into a hybrid baculovirus vector, which enhanced and prolonged H19 activation for at least 14 days in BMSC and ASC. The extended H19 activation elicited potent chondrogenic differentiation and inhibited adipogenesis. Consequently, the engineered BMSC promoted in vitro cartilage formation and augmented calvarial bone healing in rats. These data implicated the potentials of the split dCas12a activator for stem cell engineering and regenerative medicine.

Rational genome and metabolic engineering of Candida viswanathii by split CRISPR to produce hundred grams of dodecanedioic acid.

Candida viswanathii is a promising cell factory for producing dodecanedioic acid (DDA) and other long chain dicarboxylic acids. However, metabolic engineering of C. viswanathii is difficult partly due to the lack of synthetic biology toolkits. Here we developed CRISPR-based approaches for rational genome and metabolic engineering of C. viswanathii. We first optimized the CRISPR system and protocol to promote the homozygous gene integration efficiency to >60%. We also designed a split CRISPR system for one-step integration of multiple genes into C. viswanathii. We uncovered that co-expression of CYP52A19, CPRb and FAO2 that catalyze different steps in the biotransformation enhances DDA production and abolishes accumulation of intermediates. We also unveiled that co-expression of additional enzyme POS5 further promotes DDA production and augments cell growth. We harnessed the split CRISPR system to co-integrate these 4 genes (13.6 kb) into C. viswanathii and generated a stable strain that doubles the DDA titer (224 g/L), molar conversion (83%) and productivity (1.87 g/L/h) when compared with the parent strain. This study altogether identifies appropriate enzymes/promoters to augment dodecane conversion to DDA and implicates the potential of split CRISPR system for metabolic engineering of C. viswanathii.

Statistical modeling: Assessing the anatomic variability of knee joint space width.

Population-based knee joint space width (JSW) assessments are promising for the prevention and early diagnosis of osteoarthritis. This study aimed to establish the statistical shape and alignment model (SSAM) of knee joints for assessing anatomic variation in knee JSW in the healthy Chinese male population. CT scans of asymptomatic knee joints of healthy male participants (n = 107) were collected for manual segmentation to create mesh samples. The as-scanned positional error was reduced by a standard processing flow of deformable mesh registration. Principal component analysis (PCA) was performed to create a tibiofemoral SSAM that was trained on all mesh samples. The anatomic variability of the JSW in the healthy Chinese male population was then assessed using the SSAM with regression analysis and 3D analysis by color-coded mapping. Almost all PCA modes had a linear influence on the anatomic variation of the medial and lateral JSW. The JSW variability within the SSAM was mainly explained by mode 1 (45.1 % of variation), demonstrating that this mode had the greatest influence on JSW variation. 3D assessment of the JSW showed that the minimum medial JSW varied from 2.76 to 3.23 mm, and its site shifted a short distance on the medial tibial plateau. The root-mean-square fitting and generalization errors of the SSAM were below 1 mm. This study will benefit the design and optimization of prosthetic devices, and may be applicable to the prevention and early diagnosis of osteoarthritis.

Combinatorial CRISPR Interference Library for Enhancing 2,3-BDO Production and Elucidating Key Genes in Cyanobacteria.

Cyanobacteria can convert CO2 to chemicals such as 2,3-butanediol (2,3-BDO), rendering them promising for renewable production and carbon neutralization, but their applications are limited by low titers. To enhance cyanobacterial 2,3-BDO production, we developed a combinatorial CRISPR interference (CRISPRi) library strategy. We integrated the 2,3-BDO pathway genes and a CRISPRi library into the cyanobacterium PCC7942 using the orthogonal CRISPR system to overexpress pathway genes and attenuate genes that inhibit 2,3-BDO formation. The combinatorial CRISPRi library strategy allowed us to inhibit fbp, pdh, ppc, and sps (which catalyzes the synthesis of fructose-6-phosphate, acetyl-coenzyme A, oxaloacetate, and sucrose, respectively) at different levels, thereby allowing for rapid screening of a strain that enhances 2,3-BDO production by almost 2-fold to 1583.8 mg/L. Coupled with a statistical model, we elucidated that differentially inhibiting all the four genes enhances 2,3-BDO synthesis to varying degrees. fbp and pdh suppression exerted more profound effects on 2,3-BDO production than ppc and sps suppression, and these four genes can be repressed simultaneously without mutual interference. The CRISPRi library approach paves a new avenue to combinatorial metabolic engineering of cyanobacteria.

Bi-directional gene activation and repression promote ASC differentiation and enhance bone healing in osteoporotic rats.

Calvarial bone healing is challenging, especially for individuals with osteoporosis because stem cells from osteoporotic patients are highly prone to adipogenic differentiation. Based on previous findings that chondrogenic induction of adipose-derived stem cells (ASCs) can augment calvarial bone healing, we hypothesized that activating chondroinductive Sox Trio genes (Sox5, Sox6, Sox9) and repressing adipoinductive genes (C/ebp-α, Ppar-γ) in osteoporotic ASCs can reprogram cell differentiation and improve calvarial bone healing after implantation. However, simultaneous gene activation and repression in ASCs is difficult. To tackle this problem, we built a CRISPR-BiD system for bi-directional gene regulation. Specifically, we built a CRISPR-AceTran system that exploited both histone acetylation and transcription activation for synergistic Sox Trio activation. We also developed a CRISPR interference (CRISPRi) system that exploited DNA methylation for repression of adipoinductive genes. We combined CRISPR-AceTran and CRISPRi to form the CRISPR-BiD system, which harnessed three mechanisms (transcription activation, histone acetylation, and DNA methylation). After delivery into osteoporotic rat ASCs, CRISPR-BiD significantly enhanced chondrogenesis and in vitro cartilage formation. Implantation of the engineered osteoporotic ASCs into critical-sized calvarial bone defects significantly improved bone healing in osteoporotic rats. These results implicated the potential of the CRISPR-BiD system for bi-directional regulation of cell fate and regenerative medicine.

Polyplex nanomicelle delivery of self-amplifying RNA vaccine.

Self-amplifying RNA (SaRNA) is a burgeoning platform that exploits the replication machinery of alphaviruses such as Venezuelan equine encephalitis (VEE) virus or Sindbis virus (SIN). SaRNA has been used for development of human vaccines, but has not been evaluated for porcine vaccine development. Porcine reproductive and respiratory syndrome virus (PRRSV) causes tremendous economic losses to the worldwide pork industry, but current vaccines trigger delayed neutralizing antibody response and confer only partial protection. Here we first compared two SaRNA systems based on VEE and SIN, and demonstrated that in vitro transcribed VEE-based SaRNA conferred prolonged reporter gene expression and RNA amplification in pig cells with low cytotoxicity, but SIN-based SaRNA imparted evident cytotoxicity and limited gene expression in pig cells. Transfection of VEE-based SaRNA that encodes the major PRRSV antigen dNGP5 (SaRNA-dNGP5) conferred persistent expression for at least 28 days in pig cells. We next complexed SaRNA-dNGP5 with the polyaspartamide block copolymer PEG-PAsp(TEP) to form polyplex nanomicelle with high packaging efficiency and narrow size distribution. The polyplex nanomicelle enabled sustained dNGP5 expression and secretion in vitro. Compared with the commercial PRRS vaccine, nanomicelle delivery of SaRNA-dNGP5 into animal models accelerated the induction of potent neutralizing antibodies with minimal side effects, and elicited stronger IL-4 and IFN-γ responses against homologous and heterologous PRRSV. These properties tackle the problems of current vaccines and implicate the potential of SaRNA-dNGP5 nanomicelle as an effective PRRS vaccine.

CRISPR activation of long non-coding RNA DANCR promotes bone regeneration.

Healing of large calvarial bone defects in adults adopts intramembranous pathway and is difficult. Implantation of adipose-derived stem cells (ASC) that differentiate towards chondrogenic lineage can switch the bone repair pathway and improve calvarial bone healing. Long non-coding RNA DANCR was recently uncovered to promote chondrogenesis, but its roles in rat ASC (rASC) chondrogenesis and bone healing stimulation have yet to be explored. Here we first verified that DANCR expression promoted rASC chondrogenesis, thus we harnessed CRISPR activation (CRISPRa) technology to upregulate endogenous DANCR, stimulate rASC chondrogenesis and improve calvarial bone healing in rats. We generated 4 different dCas9-VPR orthologues by fusing a tripartite transcription activator domain VPR to catalytically dead Cas9 (dCas9) derived from 4 different bacteria, and compared the degree of activation using the 4 different dCas9-VPR. We unveiled surprisingly that the most commonly used dCas9-VPR derived from Streptococcus pyogenes barely activated DANCR. Nonetheless dCas9-VPR from Staphylococcus aureus (SadCas9-VPR) triggered efficient activation of DANCR in rASC. Delivery of SadCas9-VPR and the associated guide RNA into rASC substantially enhanced chondrogenic differentiation of rASC and augmented cartilage formation in vitro. Implantation of the engineered rASC remarkably potentiated the calvarial bone healing in rats. Furthermore, we identified that DANCR improved the rASC chondrogenesis through inhibition of miR-203a and miR-214. These results collectively proved that DANCR activation by SadCas9-VPR-based CRISPRa provides a novel therapeutic approach to improving calvarial bone healing.

CRISPR-Cas13d for Gene Knockdown and Engineering of CHO Cells.

Chinese hamster ovary (CHO) cells are the predominant cell chassis for biopharmaceutical production. Engineering cellular pathways related to cell death, metabolism, and glycosylation in CHO cells is desired but challenging. Here, we present a novel approach that exploits CRISPR-Cas13d for gene silencing and CHO cell engineering. CRISPR-Cas13d is a burgeoning system that exploits Cas13d nuclease and guide RNA (gRNA) for RNA cleavage and gene knockdown. We first showed that CRISPR-Cas13d effectively knocked down exogenous genes in CHO cell lines (K1, DG44, and DUXB11) commonly used for recombinant protein production. We next demonstrated that CRISPR-Cas13d robustly suppressed the expression of exogenous genes and various endogenous genes involved in gene amplification, apoptosis, metabolism, and glycosylation (e.g., GS, BAK, BAX, PDK1, and FUT8) in CHO cells with efficiencies ranging from 60% to 80%, simply by transient transfection. By integrating the entire CRISPR-Cas13d system with the Sleeping Beauty system and optimal gRNA design, we further improved the knockdown efficiency and rapidly generated stable cells with ≈80%-90% knockdown. With this approach, we knocked down FUT8 expression for >90% and significantly attenuated the IgG fucosylation. These data altogether implicated the potentials of CRISPR-Cas13d for gene regulation, glycoengineering, and cell engineering of CHO cells.

Removal of an abluminal lining improves decellularization of human umbilical arteries.

The decellularization of long segments of tubular tissues such as blood vessels may be improved by perfusing decellularization solution into their lumen. Particularly, transmural flow that may be introduced by the perfusion, if any, is beneficial to removing immunogenic cellular components in the vessel wall. When human umbilical arteries (HUAs) were perfused at a transmural pressure, however, very little transmural flow was observed. We hypothesized that a watertight lining at the abluminal surface of HUAs hampered the transmural flow and tested the hypothesis by subjecting the abluminal surface to enzyme digestion. Specifically, a highly viscous collagenase solution was applied onto the surface, thereby restricting the digestion to the surface. The localized digestion resulted in a water-permeable vessel without damaging the vessel wall. The presence of the abluminal lining and its successful removal were also supported by evidence from SEM, TEM, and mechanical testing. The collagenase-treated HUAs were decellularized with 1% sodium dodecyl sulfate (SDS) solution under either rotary agitation, simple perfusion, or pressurized perfusion. Regardless of decellularization conditions, the decellularization of HUAs was significantly enhanced after the abluminal lining removal. Particularly, complete removal of DNA was accomplished in 24 h by pressurized perfusion of the SDS solution. We conclude that the removal of the abluminal lining can improve the perfusion-assisted decellularization.

Fetal Rhabdomyoma of the Right Tonsil with Polyp-Like Appearance.

Skeletal muscle neoplasms, in contrast to other groups of tumors, are almost malignant. The benign variant, rhabdomyoma, is distinctly rare. Rhabdomyomas can be classified generally into two types: cardiac and extracardiac. Extracardiac rhabdomyoma can be further divided into three subtypes: adult, fetal, and genital type. Adult rhabdomyoma is the most common subtype of rhabdomyoma even though it remains relatively rare. Fetal rhabdomyomas are less common than the adult type. In this paper we report a rare case of a fetal rhabdomyoma with polyp-like appearance originating from right tonsil. Punch biopsy and then right tonsillectomy were performed for complete excision. There was no obvious recurrence.

Olfactory loss after uvulopalatopharyngoplasty: a report of two cases with review of the literature.

Uvulopalatopharyngoplasty (UPPP) has been a popular surgical method for treating obstructive sleep apnea syndrome since it was introduced in the early 1980s. Olfactory loss has been reported as a rare side effect in several cases. However, the olfactory test results and the prognosis were not mentioned in these cases. We present two patients who complained of loss of olfactory function after UPPP. Their olfactory function was evaluated by the phenyl ethyl alcohol odor detection threshold test and the University of Pennsylvania Smell Identification Test. After treatment with steroid and zinc salt, their olfactory function was improved but not recovered completely.

Enhancement of light scattering and photoluminescence in electrospun polymer nanofibers.

Poly(methyl methacrylate) nanofibers with desired fiber diameters that ranged from 336 to 896 nm were electrospun as light scattering and propagation materials. The light scattering behavior of these samples as a function of the fiber diameter and fiber deposition thickness was examined by UV-vis spectrophotometry, which revealed the scattering bands in the absorption spectra. The scattering bands of these nanofibers were linearly proportional to the fiber diameter, which shows good agreement with a scattering model based on the Mie theory. The light scattering and prolonged light path lengths in the nanofiber scaffolds were monitored and quantified by the photoluminescence of a fluorescent dye, Coumarin 6, which was preloaded into the polymer nanofibers. The photoluminescence after proper normalization showed a second-order dependence on the dye loading per unit area, which is significantly different from the spin-coated thin-film samples following a first-order relationship. Nonlinear photoluminescence enhancements indicated prolonged light path lengths and multiple light absorptions within the fiber scaffolds as a result of light scattering. Even with relatively broad scattering band widths, the light scattering and photoluminescence of the electrospun nanofibers exhibited considerable wavelength selectivity, especially as the scattering bands overlapped with the excitation wavelengths of the fluorescence reagent.