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.

Auto-inducible synthetic pathway in E. coli enhanced sustainable indigo production from glucose.

Indigo is widely used in textile industries for denim garments dyeing and is mainly produced by chemical synthesis which, however, raises environmental sustainability issues. Bio-indigo may be produced by fermentation of metabolically engineering bacteria, but current methods are economically incompetent due to low titer and the need for an inducer. To address these problems, we first characterized several synthetic promoters in E. coli and demonstrated the feasibility of inducer-free indigo production from tryptophan using the inducer-free promoter. We next coupled the tryptophan-to-indigo and glucose-to-tryptophan pathways to generate a de novo glucose-to-indigo pathway. By rational design and combinatorial screening, we identified the optimal promoter-gene combinations, which underscored the importance of promoter choice and expression levels of pathway genes. We thus created a new E. coli strain that exploited an indole pathway to enhance the indigo titer to 123 mg/L. We further assessed a panel of heterologous tryptophan synthase homologs and identified a plant indole lyase (TaIGL), which along with modified pathway design, improved the indigo titer to 235 mg/L while reducing the tryptophan byproduct accumulation. The optimal E. coli strain expressed 8 genes essential for rewiring carbon flux from glucose to indole and then to indigo: mFMO, ppsA, tktA, trpD, trpC, TaIGL and feedback-resistant aroG and trpE. Fed-batch fermentation in a 3-L bioreactor with glucose feeding further increased the indigo titer (≈965 mg/L) and total quantity (≈2183 mg) at 72 h. This new synthetic glucose-to-indigo pathway enables high-titer indigo production without the need of inducer and holds promise for bio-indigo production.

Benefits of Hepatitis C Viral Eradication: A Real-World Nationwide Cohort Study in Taiwan.

BACKGROUND: Chronic hepatitis C (CHC) increases the risk of liver cirrhosis (LC) and hepatocellular carcinoma (HCC). This nationwide cohort study assessed the effectiveness of viral eradication of CHC. METHODS: The Taiwanese chronic hepatitis C cohort and Taiwan hepatitis C virus (HCV) registry are nationwide HCV registry cohorts incorporating data from 23 and 53 hospitals in Taiwan, respectively. This study included 27,577 individuals from these cohorts that were given a diagnosis of CHC and with data linked to the Taiwan National Health Insurance Research Database. Patients received either pegylated interferon and ribavirin or direct-acting antiviral agent therapy for > 4 weeks for new-onset LC and liver-related events. RESULTS: Among the 27,577 analyzed patients, 25,461 (92.3%) achieved sustained virologic response (SVR). The mean follow-up duration was 51.2 ± 48.4 months, totaling 118,567 person-years. In the multivariable Cox proportional hazard analysis, the hazard ratio (HR) for incident HCC was 1.39 (95% confidence interval [CI]: 1.00-1.95, p = 0.052) among noncirrhotic patients without SVR compared with those with SVR and 1.82 (95% CI 1.34-2.48) among cirrhotic patients without SVR. The HR for liver-related events, including HCC and decompensated LC, was 1.70 (95% CI 1.30-2.24) among cirrhotic patients without SVR. Patients with SVR had a lower 10-year cumulative incidence of new-onset HCC than those without SVR did (21.7 vs. 38.7% in patients with LC, p < 0.001; 6.0 vs. 18.4% in patients without LC, p < 0.001). CONCLUSION: HCV eradication reduced the incidence of HCC in patients with and without LC and reduced the incidence of liver-related events in patients with LC.

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.

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.

Chinese Intelligence Prescription System improves prescription accuracy while decreasing labor and drug costs.

BACKGROUND AND AIM: The traditional method of taking Chinese Medicine involves creating a decoction by cooking medicinal Chinese herbs. However, this method has become less popular, being replaced by the more convenient method of consuming concentrated Chinese herbal extracts, which creates challenges related to the complexity of stacking multiple formulas. METHODS: We developed the Chinese Intelligence Prescription System (CIPS) to simplify the prescription process. In this study, we used data from our institutions pharmacy to calculate the number of reductions, average dispensing time, and resulting cost savings. RESULTS: The mean number of prescriptions was reduced from 8.19 ± 3.65 to 7.37 ± 3.34 ([Formula: see text]). The reduction in the number of prescriptions directly resulted in decreased dispensing time, reducing it from 1.79 ± 0.25 to 1.63 ± 0.66 min ([Formula: see text]). The reduced dispensing time totaled 3.75 h per month per pharmacist, equivalent to an annual labor cost savings of $15,488 NTD per pharmacist. In addition, drug loss was reduced during the prescription process, with a mean savings of $4,517 NTD per year. The combined savings adds up to a not insignificant $20,005 NTD per year per pharmacist. When taking all TCM clinics/hospitals in Taiwan into account, the total annual savings would be $77 million NTD. CONCLUSION: CIPS assists clinicians and pharmacists to formulate precise prescriptions in a clinical setting to simplify the dispensing process while reducing medical resource waste and labor costs.

Synthetic switch to minimize CRISPR off-target effects by self-restricting Cas9 transcription and translation.

CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of cas9 gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel 'hit and run' strategy for in vivo genome editing.

Alignment-Free Wireless Charging of Smart Garments with Embroidered Coils.

Wireless power transfer (WPT) technologies have been adopted by many products. The capability of charging multiple devices and the design flexibility of charging coils make WPT a good solution for charging smart garments. The use of an embroidered receiver (RX) coil makes the smart garment more breathable and comfortable than using a flexible printed circuit board (FPCB). In order to charge smart garments as part of normal daily routines, two types of wireless-charging systems operating at 400 kHz have been designed. The one-to-one hanger system is desired to have a constant charging current despite misalignment so that users do not need to pay much attention when they hang the garment. For the one-to-multiple-drawer system, the power delivery ability must not change with multiple garments. Additionally, the system should be able to charge folded garments in most of the folding scenarios. This paper analyses the two WPT systems for charging smart garments and provides design approaches to meet the abovementioned goals. The wireless-charging hanger is able to charge a smart garment over a coupling variance kmaxkmin=2 with only 21% charging current variation. The wireless-charging drawer is able to charge a smart garment with at least 20 mA under most folding scenarios and three garments with stable power delivery ability.

Synthetic switch-based baculovirus for transgene expression control and selective killing of hepatocellular carcinoma cells.

Baculovirus (BV) holds promise as a vector for anticancer gene delivery to combat the most common liver cancer-hepatocellular carcinoma (HCC). However, in vivo BV administration inevitably results in BV entry into non-HCC normal cells, leaky anticancer gene expression and possible toxicity. To improve the safety, we employed synthetic biology to engineer BV for transgene expression regulation. We first uncovered that miR-196a and miR-126 are exclusively expressed in HCC and normal cells, respectively, which allowed us to engineer a sensor based on distinct miRNA expression signature. We next assembled a synthetic switch by coupling the miRNA sensor and RNA binding protein L7Ae for translational repression, and incorporated the entire device into a single BV. The recombinant BV efficiently entered HCC and normal cells and enabled cis-acting transgene expression control, by turning OFF transgene expression in normal cells while switching ON transgene expression in HCC cells. Using pro-apoptotic hBax as the transgene, the switch-based BV selectively killed HCC cells in separate culture and mixed culture of HCC and normal cells. These data demonstrate the potential of synthetic switch-based BV to distinguish HCC and non-HCC normal cells for selective transgene expression control and killing of HCC cells.

RNA-guided genome engineering: paradigm shift towards transposons.

CRISPR-Cas systems revolutionized the genome engineering field but need to induce double-strand breaks (DSBs) and may be difficult to deliver due to their large protein size. Tn7-like transposons such as CRISPR-associated transposons (CASTs) can be repurposed for RNA-guided DSB-free integration, and obligate mobile element guided activity (OMEGA) proteins of the IS200/IS605 transposon family have been developed as hypercompact RNA-guided genome editing tools. CASTs and OMEGA are exciting, innovative genome engineering tools that can improve the precision and efficiency of editing. This review explores the recent developments and uses of CASTs and OMEGA in genome editing across prokaryotic and eukaryotic cells. The pros and cons of these transposon-based systems are deliberated in comparison to other CRISPR systems.

Programmable editing of primary MicroRNA switches stem cell differentiation and improves tissue regeneration.

Programmable RNA editing is harnessed for modifying mRNA. Besides mRNA, miRNA also regulates numerous biological activities, but current RNA editors have yet to be exploited for miRNA manipulation. To engineer primary miRNA (pri-miRNA), the miRNA precursor, we present a customizable editor REPRESS (RNA Editing of Pri-miRNA for Efficient Suppression of miRNA) and characterize critical parameters. The optimized REPRESS is distinct from other mRNA editing tools in design rationale, hence enabling editing of pri-miRNAs that are not editable by other RNA editing systems. We edit various pri-miRNAs in different cells including adipose-derived stem cells (ASCs), hence attenuating mature miRNA levels without disturbing host gene expression. We further develop an improved REPRESS (iREPRESS) that enhances and prolongs pri-miR-21 editing for at least 10 days, with minimal perturbation of transcriptome and miRNAome. iREPRESS reprograms ASCs differentiation, promotes in vitro cartilage formation and augments calvarial bone regeneration in rats, thus implicating its potentials for engineering miRNA and applications such as stem cell reprogramming and tissue regeneration.

Prescription system to calculate precise doses of Chinese herbal medicine to avoid toxic effects.

Background and objectives: Traditional Chinese Medicine (TCM) is a therapeutic system which has been practiced for thousands of years. Although for much of its history the decoction of medicinal herbs was the most common method of consuming the herbal treatments, TCM prescriptions are now primarily prepared using concentrated Chinese herbal extracts (CCHE) in powder or granular form. However, determining the precise dose of each single Chinese herbal constituent within a prescription creates a challenge in clinical practice due to the potential risk of toxicity. To alleviate this, we invented the Chinese Intelligence Prescription System (CIPS) to calculate the exact dose of each single herb within an individual prescription. Methods: In this study, we applied CIPS in a real-world setting to analyze clinical prescriptions collected and prepared at the TCM Pharmacy of China Medical University Hospital (CMUH). Results: Our investigation revealed that 3% of all prescriptions filled in a 1-month period contained inexact dosages, suggesting that more than 170,000 prescriptions filled in Taiwan in a given month may contain potentially toxic components. We further analyzed the data to determine the excess dosages and outline the possible associated side effects. Conclusions: In conclusion, CIPS offers TCM practitioners the ability to prepare exact Chinese herbal medicine (CHM) prescriptions in order to avoid toxic effects, thereby ensuring patient safety.

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.

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.

Rapid SARS-CoV-2 diagnosis using disposable strips and a metal-oxide-semiconductor field-effect transistor platform.

The SARS-CoV-2 pandemic has had a significant impact worldwide. Currently, the most common detection methods for the virus are polymerase chain reaction (PCR) and lateral flow tests. PCR takes more than an hour to obtain the results and lateral flow tests have difficulty with detecting the virus at low concentrations. In this study, 60 clinical human saliva samples, which included 30 positive and 30 negative samples confirmed with RT-PCR, were screened for COVID-19 using disposable glucose biosensor strips and a reusable printed circuit board. The disposable strips were gold plated and functionalized to immobilize antibodies on the gold film. After functionalization, the strips were connected to the gate electrode of a metal-oxide-semiconductor field-effect transistor on the printed circuit board to amplify the test signals. A synchronous double-pulsed bias voltage was applied to the drain of the transistor and strips. The resulting change in drain waveforms was converted to digital readings. The RT-PCR-confirmed saliva samples were tested again using quantitative PCR (RT-qPCR) to determine cycling threshold (Ct) values. Ct values up to 45 refer to the number of amplification cycles needed to detect the presence of the virus. These PCR results were compared with digital readings from the sensor to better evaluate the sensor technology. The results indicate that the samples with a range of Ct values from 17.8 to 35 can be differentiated, which highlights the increased sensitivity of this sensor technology. This research exhibits the potential of this biosensor technology to be further developed into a cost-effective, point-of-care, and portable rapid detection method for SARS-CoV-2.

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.

Fast SARS-CoV-2 virus detection using disposable cartridge strips and a semiconductor-based biosensor platform.

Detection of the SARS-CoV-2 spike protein and inactivated virus was achieved using disposable and biofunctionalized functional strips, which can be connected externally to a reusable printed circuit board for signal amplification with an embedded metal-oxide-semiconductor field-effect transistor (MOSFET). A series of chemical reactions was performed to immobilize both a monoclonal antibody and a polyclonal antibody onto the Au-plated electrode used as the sensing surface. An important step in the biofunctionalization, namely, the formation of Au-plated clusters on the sensor strips, was verified by scanning electron microscopy, as well as electrical measurements, to confirm successful binding of thiol groups on this Au surface. The functionalized sensor was externally connected to the gate electrode of the MOSFET, and synchronous pulses were applied to both the sensing strip and the drain contact of the MOSFET. The resulting changes in the dynamics of drain waveforms were converted into analog voltages and digital readouts, which correlate with the concentration of proteins and virus present in the tested solution. A broad range of protein concentrations from 1 fg/ml to 10 µg/ml and virus concentrations from 100 to 2500 PFU/ml were detectable for the sensor functionalized with both antibodies. The results show the potential of this approach for the development of a portable, low-cost, and disposable cartridge sensor system for point-of-care detection of viral diseases.

Design-based guidelines for the semantic perception of emergency signs.

The current study applies semantic differential to explore the semantic perception of emergency signs, in an attempt to analyze the meanings of emergency signs in regard to the psychological exigencies of the general public. The results indicate that problems concerning recognition accuracy have been observed, but also that the evaluation of the semantic meaning of the signs is positive. However, a number of emotional factors also emerge, indicating that if improvements should be made to the existing emergency signs, it would better facilitate evacuation in the event of an emergency. As for the design guidelines, the emergency signs should be readily recognizable and have a single meaning. Multiple symbolic meanings should be eliminated. Concrete design guidelines include: (a) safety movement, (b) clarity, and (c) safety. To avoid confusion in sign recognition, in addition to the improvements made based on the abovementioned guidelines, educational promotional activities carried out by the government would also prove very beneficial.

A Two-Electrode, Double-Pulsed Sensor Readout Circuit for Cardiac Troponin I Measurement.

This paper presents a pulse-stimulus sensor readout circuit for use in cardiovascular disease examinations. The sensor is based on a gold nanoparticle plate with an antibody post-modification. The proposed system utilizes gated pulses to detect the biomarker Cardiac Troponin I in an ionic solution. The characteristic of the electrostatic double-layer capacitor generated by the analyte is related to the concentration of Cardiac Troponin I in the solvent. After sensing by the transistor, a current-to-frequency converter (I-to-F) and delay-line-based time-to-digital converter (TDC) convert the information into a series of digital codes for further analysis. The design is fabricated in a 0.18-µm standard CMOS process. The chip occupies an area of 0.92 mm2 and consumes 125 µW. In the measurements, the proposed circuit achieved a 1.77 Hz/pg-mL sensitivity and 72.43 dB dynamic range.

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.