An engineered Pichia pastoris platform for the biosynthesis of silk-based nanomaterials with therapeutic potential.

Silk fibroin (SF) from the cocoon of silkworm has exceptional mechanical properties and biocompatibility and is used as a biomaterial in a variety of fields. Sustainable, affordable, and scalable manufacturing of SF would enable its large-scale use. We report for the first time the high-level secretory production of recombinant SF peptides in engineered Pichia pastoris cell factories and the processing thereof to nanomaterials. Two SF peptides (BmSPR3 and BmSPR4) were synthesized and secreted by P. pastoris using signal peptides and appropriate spacing between hydrophilic sequences. By strain engineering to reduce protein degradation, increase glycyl-tRNA supply, and improve protein secretion, we created the optimized P. pastoris chassis PPGSP-8 to produce BmSPR3 and BmSPR4. The SF fed-batch fermentation titers of the resulting two P. pastoris cell factories were 11.39 and 9.48 g/L, respectively. Protein self-assembly was inhibited by adding Tween 80 to the medium. Recombinant SF peptides were processed to nanoparticles (NPs) and nanofibrils. The physicochemical properties of nanoparticles R3NPs and R4NPs from the recombinant SFs synthesized in P. pastoris cell factories were similar or superior to those of RSFNPs (Regenerated Silk Fibroin NanoParticles) originating from commercially available SF. Our work will facilitate the production by microbial fermentation of functional SF for use as a biomaterial.

The protein methyltransferase TrSAM inhibits cellulase gene expression by interacting with the negative regulator ACE1 in Trichoderma reesei.

Protein methylation is a commonly posttranslational modification of transcriptional regulators to fine-tune protein function, however, whether this regulation strategy participates in the regulation of lignocellulase synthesis and secretion in Trichoderma reesei remains unexplored. Here, a putative protein methyltransferase (TrSAM) is screened from a T. reesei mutant with the ability to express heterologous ß-glucosidase efficiently even under glucose repression. The deletion of its encoding gene trsam causes a significant increase of cellulase activities in all tested T. reesei strains, including transformants of expressing heterologous genes using cbh1 promotor. Further investigation confirms that TrSAM interacts with the cellulase negative regulator ACE1 via its amino acid residue Arg383, which causes a decrease in the ACE1-DNA binding affinity. The enzyme activity of a T. reesei strain harboring ACE1R383Q increases by 85.8%, whereas that of the strains with trsam or ace1 deletion increases by more than 100%. By contrast, the strain with ACE1R383K shows no difference to the parent strain. Taken together, our results demonstrate that TrSAM plays an important role in regulating the expression of cellulase and heterologous proteins initiated by cbh1 promotor through interacting with ACE1R383. Elimination and mutation of TrSAM and its downstream ACE1 alleviate the carbon catabolite repression (CCR) in expressing cellulase and heterologous protein in varying degrees. This provides a new solution for the exquisite modification of T. reesei chassis.

The Impact of Australian Bushfires on Asthma Medicine Prescription Dispensing.

BACKGROUND: Air pollution can cause numerous health problems and increase the need for medicines to treat and prevent asthma in affected areas. There is limited evidence about the association between airborne particles with a diameter of 2.5 micrometres or smaller (PM2.5) and asthma medicine usage. This study examined the potential association between the levels of PM2.5 and the supply of prescription asthma medicines in the Australian Capital Territory (ACT), Australia, during the severe bushfire season between November 2019-January 2020. METHODS: Daily data was obtained from an ACT air quality monitoring station from November 2019 to January 2020 (study period) and November 2018 to January 2019 (control period, no bushfire). The number and types of government-funded asthma medicine prescriptions were obtained from the Services Australia (government) website by searching under 'Pharmaceutical Benefits Scheme Item Reports' and using relevant item codes during the study and control periods. RESULTS: The medians for PM2.5 levels for the study period were significantly higher than those for the control period (p < 0.001). There were increases in the number of dispensed prescriptions of short-acting beta-2 agonists (SABA), inhaled corticosteroids, and long-acting beta-2 agonists combined with inhaled corticosteroids. The greatest difference was seen with the inhaled corticosteroids: a 138% increase. CONCLUSIONS: The increase in the number of dispensed asthma prescriptions during the bushfire season should be used to inform the stock holdings of these medicines in preparation for future events to ensure access to lifesaving asthma medicines.

Development and validation of prognostic nomogram for T1-3N0M0 non-small cell lung cancer after curative resection.

BACKGROUND: Radical resection plus lymph node dissection is a common treatment for patients with T1-3N0M0 non-small cell lung cancer (NSCLC). Few models predicted the survival outcomes of these patients. This study aimed to developed a nomogram for predicting their overall survival (OS). MATERIALS AND METHODS: This study involved 3002 patients with T1-3N0M0 NSCLC after curative resection between January 1999 and October 2013. 1525 Patients from Sun Yat-sen University Cancer Center were randomly allocated to training cohort and internal validation cohort in a ratio of 7:3. 1477 patients from ten institutions were recruited as external validation cohort. A nomogram was constructed based on the training cohort and validated by internal and external validation cohort to predict the OS of these patients. The accuracy and practicability were tested by Harrell's C-indexes, calibration plots and decision curve analyses (DCA). RESULTS: Age, sex, histological classification, pathological T stage, and HI standard were independent factors for OS and were included in our nomogram. The C-index of the nomogram for OS estimates were 0.671 (95% CI, 0.637-0.705),0.632 (95% CI, 0.581-0.683), and 0.645 (95% CI, 0.617-0.673) in the training cohorts, internal validation cohorts, and external validation cohort, respectively. The calibration plots and DCA for predictions of OS were in excellent agreement. An online version of the nomogram was built for convenient clinical practice. CONCLUSIONS: Our nomogram can predict the OS of patients with T1-3N0M0 NSCLC after curative resection. The online version of our nomogram offer opportunities for fast personalized risk stratification and prognosis prediction in clinical practice.

The computational rules of cross-modality suppression in the visual posterior sylvian area.

The macaque visual posterior sylvian area (VPS) is an area with neurons responding selectively to heading direction in both visual and vestibular modalities, but how VPS neurons combined these two sensory signals is still unknown. In contrast to the subadditive characteristics in the medial superior temporal area (MSTd), responses in VPS were dominated by vestibular signals, with approximately a winner-take-all competition. The conditional Fisher information analysis shows that VPS neural population encodes information from distinct sensory modalities under large and small offset conditions, which differs from MSTd whose neural population contains more information about visual stimuli in both conditions. However, the combined responses of single neurons in both areas can be well fit by weighted linear sums of unimodal responses. Furthermore, a normalization model captured most vestibular and visual interaction characteristics for both VPS and MSTd, indicating the divisive normalization mechanism widely exists in the cortex.

Emamectin benzoate exposure impaired porcine oocyte maturation.

Emamectin benzoate (EB) is a widely used insecticide that can damage the central nervous and immune systems. EB exposure significantly reduced the number of eggs laid, hatching rate, and developmental rate of lower organisms such as nematodes. However, effects of EB exposure on the maturation of higher animals such as porcine oocytes remains unknown. Here we reported that EB exposure severely impaired porcine oocyte maturation. EB exposure with 200 µM prevented cumulus expansion and reduced the rates of first polar body (pb1) extrusion, cleavage and blastocyst after parthenogenetic activation. Moreover, EB exposure disrupted spindle organization, chromosome alignment, and polymerization of microfilaments, but also apparently decreased the levels of acetylated α-tubulin (Ac-Tub) in oocytes. In addition, EB exposure perturbed mitochondria distribution and increased levels of reactive oxygen species (ROS), but did not affect the distribution of cortical granules (CGs) in oocytes. Excessive ROS caused DNA damage accumulation and induced early apoptosis of oocytes. EB exposure led to the abnormal expression of cumulus expansion and apoptosis-associated genes. Altogether, these results demonstrate that EB exposure impaired nuclear and cytoplasmic maturation of porcine oocytes probably through oxidative stress and early apoptosis.

Inhibitory Effects of 3-Methylcholanthrene Exposure on Porcine Oocyte Maturation.

3-methylcholanthrene (3-MC) is a highly toxic environmental pollutant that impairs animal health. 3-MC exposure can cause abnormal spermatogenesis and ovarian dysfunction. However, the effects of 3-MC exposure on oocyte maturation and embryo development remain unclear. This study revealed the toxic effects of 3-MC exposure on oocyte maturation and embryo development. 3-MC with different concentrations of 0, 25, 50, and 100 µM was applied for in vitro maturation of porcine oocytes. The results showed that 100 µM 3-MC significantly inhibited cumulus expansion and the first polar body extrusion. The rates of cleavage and blastocyst of embryos derived from 3-MC-exposed oocytes were significantly lower than those in the control group. Additionally, the rates of spindle abnormalities and chromosomal misalignments were higher than those in the control group. Furthermore, 3-MC exposure not only decreased the levels of mitochondria, cortical granules (CGs), and acetylated α-Tubulin, but also increased the levels of reactive oxygen species (ROS), DNA damage, and apoptosis. The expression of cumulus expansion and apoptosis-related genes was abnormal in 3-MC-exposed oocytes. In conclusion, 3-MC exposure disrupted the nuclear and cytoplasmic maturation of porcine oocytes through oxidative stress.

Exposure-response relationship between temperature, relative humidity, and varicella: a multicity study in South China.

Varicella is a rising public health issue. Several studies have tried to quantify the relationships between meteorological factors and varicella incidence but with inconsistent results. We aim to investigate the impact of temperature and relative humidity on varicella, and to further explore the effect modification of these relationships. In this study, the data of varicella and meteorological factors from 2011 to 2019 in 21 cities of Guangdong Province, China were collected. Distributed lag nonlinear models (DLNM) were constructed to explore the relationship between meteorological factors (temperature and relative humidity) and varicella in each city, controlling in school terms, holidays, seasonality, long-term trends, and day of week. Multivariate meta-analysis was applied to pool the city-specific estimations. And the meta-regression was used to explore the effect modification for the spatial heterogeneity of city-specific meteorological factors and social factors (such as disposable income per capita, vaccination coverage, and so on) on varicella. The results indicated that the relationship between temperature and varicella in 21 cities appeared nonlinear with an inverted S-shaped. The relative risk peaked at 20.8 ℃ (RR = 1.42, 95% CI: 1.22, 1.65). The relative humidity-varicella relationship was approximately L-shaped, with a peaking risk at 69.5% relative humidity (RR = 1.25, 95% CI: 1.04, 1.50). The spatial heterogeneity of temperature-varicella relationships may be caused by income or varicella vaccination coverage. And varicella vaccination coverage may contribute to the spatial heterogeneity of the relative humidity-varicella relationship. The findings can help us deepen the understanding of the meteorological factors-varicella association and provide evidence for developing prevention strategy for varicella epidemic.

RankAxis: Towards a Systematic Combination of Projection and Ranking in Multi-Attribute Data Exploration.

Projection and ranking are frequently used analysis techniques in multi-attribute data exploration. Both families of techniques help analysts with tasks such as identifying similarities between observations and determining ordered subgroups, and have shown good performances in multi-attribute data exploration. However, they often exhibit problems such as distorted projection layouts, obscure semantic interpretations, and non-intuitive effects produced by selecting a subset of (weighted) attributes. Moreover, few studies have attempted to combine projection and ranking into the same exploration space to complement each other's strengths and weaknesses. For this reason, we propose RankAxis, a visual analytics system that systematically combines projection and ranking to facilitate the mutual interpretation of these two techniques and jointly support multi-attribute data exploration. A real-world case study, expert feedback, and a user study demonstrate the efficacy of RankAxis.

Assessing the impact of COVID-19 interventions on the hand, foot and mouth disease in Guangdong Province, China: a Bayesian modeling study.

Background: The non-pharmaceutical interventions (NPIs) against COVID-19 may have affected the transmission of hand, foot and mouth disease (HFMD). We aimed to assess the impact of the NPIs on HFMD in the high epidemic area of HFMD, Guangdong Province. Methods: The data of HFMD cases, etiological information, and meteorological factors in Guangdong from January 1, 2012, to December 31, 2021, were collected. Using a Bayesian structural time series (BSTS) model integrated counterfactual framework, we assessed the effect of NPIs on HFMD by different intervention periods, populations (gender, age, occupation), and cities. We further explored the correlation between the reduction of HFMD and socioeconomic factors in 21 cities. Results: A total of 351,217 HFMD cases were reported and 455,327 cases were averted in Guangdong Province during 2020-2021 with a reduction of 84.94% (95%CI: 81.63-87.22%) in 2020 and 29.49% (95%CI: 15.26-39.54%) in 2021. The impact of NPIs on HFMD differed by age and gender. The effects of NPIs were more remarkable for children aged 0-2 years and scattered children. We found that the relative reductions in 21 cities were related to the composition ratio of children and COVID-19 incidence. Conclusion: The reduction of HFMD incidence was significantly associated with COVID-19 NPIs, and school closure was an effective intervention to prevent HFMD outbreaks. Our findings will contribute to the development of HFMD prevention and control measures.

Spatial-temporal clustering of an outbreak of SARS-CoV-2 Delta VOC in Guangzhou, China in 2021.

Background: In May 2021, the SARS-CoV-2 Delta variant led to the first local outbreak in China in Guangzhou City. We explored the epidemiological characteristics and spatial-temporal clustering of this outbreak. Methods: Based on the 153 cases in the SARS-CoV-2 Delta variant outbreak, the Knox test was used to analyze the spatial-temporal clustering of the outbreak. We further explored the spatial-temporal clustering by gender and age groups, as well as compared the changes of clustering strength (S) value between the two outbreaks in Guangzhou. Results: The result of the Knox analysis showed that the areas at short distances and brief periods presented a relatively high risk. The strength of clustering of male-male pairs was higher. Age groups showed that clustering was concentrated in cases aged ≤ 18 years matched to 18-59 years and cases aged 60+ years. The strength of clustering of the outbreak declined after the implementation of public health measures. The change of strength of clustering at time intervals of 1-5 days decreased greater in 2021 (S = 129.19, change rate 38.87%) than that in 2020 (S = 83.81, change rate 30.02%). Conclusions: The outbreak of SARS-CoV-2 Delta VOC in Guangzhou has obvious spatial-temporal clustering. The timely intervention measures are essential role to contain this outbreak of high transmission.

Expression Profiling of Circular RNAs in Early Pregnant Jianghuai Buffaloes.

Circular RNA (circRNA) is expressed in cells and tissues of several species. However, the expression of circRNAs in the blood of Jianghuai buffaloes during early pregnancy has not been reported. In this study, we identified the DECs in the blood of Jianghuai buffaloes and annotated the functions of these DECs. The results showed that there were 890 DECs between the pregnant and non-pregnant groups, of which more than 80% were exon-derived circRNAs, including 323 up-regulated circRNAs and 567 down-regulated circRNAs. Enrichment analysis revealed that DECs were mainly enriched in the epidermal growth factor receptor-signaling pathway important for embryonic development and pregnancy maintenance. In addition, most DECs have multiple miRNA targets, suggesting that these DECs have the potential to function as miRNA sponges. In conclusion, several DECs are present between pregnant and non-pregnant Jianghuai buffaloes, and these DECs are associated with embryo implantation and pregnancy establishment.

Controllable manipulation of alginate-gelatin core-shell microcarriers for HUMSCs expansion.

Human umbilical cord mesenchymal stem cells (HUMSCs) are one of the most attractive sources of stem cells, and it is meaningful to design and develop a type of microcarriers with suitable mechanical strength for HUMSCs proliferation in order to acquire enough cells for cell-based therapy. Alginate-gelatin core-shell (AG) soft microcarriers were thus fabricated via a microfluidic device with droplet shearing/gelation facilities and surface coating for in vitro expansion of HUMSCs. The attachment and proliferation of HUMSCs on AG microcarriers with different mechanical strengths modulated by gelatin coating was studied, and the harvested cells were characterized to verity their differentiation potential. The obtained core-shell microcarriers were all uniform in size with a high mono-dispersity (CV < 5 %). An increase in the gelatin surface coating concentration from 0.5 % to 1.5 % would lead to the reduction in both the particle size of the microcarriers and swelling ratio upon the contact of culture medium, but increased elastic modulus. Microcarriers of 245.12 µm with a gelatin coating elastic modulus of 27.5 kPa (AG10) were found to be the optimal substrate for HUMSCs with an initial attachment efficiency of 44.41 % and a 5-day expansion efficiency of 647 %. The cells harvested from AG10 still reserved their outstanding pluripotency. Fresh AG10 could smoothly transfer cells from a running microcarrier-cell system of confluence to serve as a convenient way of scaling-up the existing culture. The current study thus developed suitable microcarriers, AG10, for in vitro HUMSCs expansion with well reserve of cell multipotency, and also provided a manufacturing and surface manipulating strategy of precise production and fine regulation of microcarrier properties.

Efficient removal of sixteen priority polycyclic aromatic hydrocarbons from textile dyeing sludge using electrochemical Fe2+-activated peroxymonosulfate oxidation-A green pretreatment strategy for textile dyeing sludge toxicity reduction.

It is urgent to remove polycyclic aromatic hydrocarbons (PAHs) from textile dyeing sludge (TDS) before its final deposal due to their recalcitrant nature and generation of toxic byproducts during TDS treatment. In this study, an electrochemical Fe2+-activated peroxymonosulfate (PMS) oxidation process for removing 16 priority PAHs from real TDS was firstly investigated. The results showed that the removal efficiency of the ∑16PAHs in TDS was positively correlated to the concentration of Fe2+ released from sacrificial iron anode and the concentration of electroregenerated Fe2+ in the cathode by the reduction of Fe3+ within the applied voltage range of 3-7 V, but a higher voltage of 10 V did not lead to further improvement in ∑16PAHs removal due to the radical scavenging reaction resulted from the excessive accumulation of Fe2+. 64.7% and 16.1% of the ∑16PAHs were removed in the anodic and cathodic chamber under the optimum reaction conditions of 400 mg/g PMS/VSS, pH 3 and applied voltage 7 V, respectively. low-ring PAHs were preferentially degraded compared to high-ring PAHs. The O⋅Hplayed a major role while SO4⋅-had a minor role in PAHs degradation in TDS. The intracellular PAHs released from cracked sludge cells were found to undergo further degradation under free radical attack.

The impact of enterovirus A71 vaccination program on hand, foot, and mouth disease in Guangdong, China: A longitudinal surveillance study.

Enterovirus A71 (EV71) vaccination program was introduced in 2016 in China. Based on a longitudinal surveillance dataset from 2012 to 2019 in Guangdong, China, we estimated the impact of the EV71 vaccination program on hand, foot, and mouth disease (HFMD) incidence, by using a counterfactual prediction made from synthetic control approach integrated with a Bayesian time-series model. We observed a relative reduction of 41.4% for EV71-associated HFMD cases during the post-vaccination period of 2017-2019, corresponding to 26,226 cases averted. The reduction of EV71-associated HFMD cases raised with the elevation of EV71 vaccine coverage by year. We found an indirect effect for the children aged 6-14 years who were less likely to be vaccinated. Whereas, the EV71 vaccine may not protect against non-EV71-associated HFMD. This study provides a template for ongoing public health surveillance of EV71 vaccine effectiveness with a counterfactual study design. Our results show strong evidence of the EV71 vaccination program working on reducing EV71-associated HFMD in real-world settings. The finding will benefit policy-making of EV71 vaccination and the prevention of HFMD.

The effect of polluting cooking fuels on depression among older adults in six low- and middle-income countries.

BACKGROUND: Little is known about the association between polluting cooking fuel and depression among older adults living in low- and middle-income countries (LMICs). OBJECTIVE: To evaluate the association between polluting cooking fuel and depression in older population of LMICs. METHODS: We derived data from WHO Study on global AGEing and adult health (SAGE), which was conducted in six LMICs including China, India, Ghana, South Africa, Mexico and Russia. We applied logistic regression with the propensity score method to examine the relationship of polluting cooking fuel and depression among adults ≥50 years old. RESULTS: Overall, the odds ratio (OR) of depression was 1.57 [95% confidence intervals (CIs), 1.26-1.93] for older adults relying on polluting cooking fuel in six LMICs. In India and China, polluting cooking fuel was associated with depression with ORs of 2.06 (95%CI, 1.44-2.94) and 1.59 (95%CI, 1.01-2.49), respectively. Positive OR of depression was identified for those polluting cooking fuel users aged over 65 years old (OR, 1.65; 95%CI,1.16-2.36) and those aged 50-65 (OR, 1.50; 95%CI,1.14-1.97). Polluting cooking fuel was associated with depression for females (OR, 1.80; 95%CI, 1.32-2.46), however we did not observe significant association for males. Positive effect of polluting cooking fuel was identified in both rural (OR, 1.72; 95%CI, 1.26-2.34) and urban areas (OR, 1.44; 95%CI, 1.07-1.95). For individuals relying on solid fuel, cooking in a room used for living/sleeping and using open stove/fire was associated with depression with ORs of 1.30 (95%CI, 1.14-1.48) and 1.15 (95%CI, 1.01-1.31), respectively. However, no significant effect was identified for hood. CONCLUSION: Polluting cooking fuel was related to depressive symptoms among older adults in LMICs. In addition, cooking ventilation could be useful intervention to control health hazard of solid fuel.

The evolution of land policies in China from 1980 to 2019: a policy-text based analysis.

Land resources are indispensable for the development of a country, especially for a country such as China with a very high population relative to its land area. Since reforms were introduced and liberalization was adopted, many policies have been promulgated for the protection and utilization of land resources. In the present study, more than 2000 land policies that have been formulated and promulgated in China from 1980 to 2019 were examined and statistically analyzed to qualitatively and quantitatively introduce the evolution of these policies in China. This paper mainly studies land policy of China from three perspectives as follows: policy intensity, policy tools, and policy targets, and three principal observations emerged from these investigations. First, during the past 40 years, the quantity of land policies introduced showed an increasing trend, while policy intensity became increasingly stabilized. Second, compared with regulatory policies and stimulative policies, it seems that China prefers to adopt guiding policies to manage land resources. Third, the main purpose of land policies issued during 1980-2019 is land market regulation, with subordinate policies for land resources protection.

When and How to Adjust Non-Pharmacological Interventions Concurrent with Booster Vaccinations Against COVID-19 - Guangdong, China, 2022.

Introduction: With the large-scale roll-out of the coronavirus disease 2019 (COVID-19) booster vaccination effort (a vaccine dose given 6 months after completing primary vaccination) in China, we explore when and how China could lift non-pharmacological interventions (NPIs) against COVID-19 in 2022. Methods: Using a modified susceptible-infectious-recovered (SIR) mathematical model, we projected the COVID-19 epidemic situation and required medical resources in Guangdong Province, China. Results: If the number of people entering from overseas recovers to 20% of the number in 2019, the epidemic in 2022 could be controlled at a low level by a containment (215 local cases) or suppression strategy (1,397 local cases). A mitigation strategy would lead to 21,722 local cases. A coexistence strategy would lead to a large epidemic with 6,850,083 local cases that would overwhelm Guangdong's medical system. With 50% or 100% recovery of the 2019 level of travelers from overseas, the epidemic could also be controlled with containment or suppression, but enormous resources, including more hotel rooms for border quarantine, will be required. However, coexistence would lead to an uncontrollable epidemic with 12,922,032 local cases. Discussion: With booster vaccinations, the number of travelers from overseas could increase slightly in 2022, but a suppression strategy would need to be maintained to ensure a controllable epidemic.

Efficient genome editing in Claviceps purpurea using a CRISPR/Cas9 ribonucleoprotein method.

Claviceps purpurea produces many pharmacologically important ergot alkaloids (EAS), which are widely used to treat migraine and hypertension and to aid childbirth. Although an EAS biosynthetic cluster of C. purpurea has been discovered more than 20 years ago, the complete biosynthetic pathway of EAS has not been fully characterized until now. The main obstacle to elucidating this pathway and strain modification is the lack of efficient genome-editing tools for C. purpurea. The conventional gene manipulation method for C. purpurea relies on homologous recombination (HR), although the efficiency of HR in C. purpurea is very low (∼1-5%). Consequently, the disruption of target genes is laborious and time-consuming. Although CRISPR/Cas9 genome-editing methods based on in vivo Cas9 expression and gRNA transcription have been reported recently, their gene-disruption efficiency is still very low. Here, we developed an efficient genome-editing system in C. purpurea based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes. As proof of principle, three target genes were efficiently knocked out using this CRISPR/Cas9 ribonucleoprotein complex-mediated HR system, with editing efficiencies ranging from 50% to 100%. Inactivation of the three genes, which are closely related to uridine biosynthesis (ura5), hypha morphology (rac), and EAS production (easA), resulted in a uridine auxotrophic mutant, a mutant with a drastically different phenotype in axenic culture, and a mutant that did not produce EAS, respectively. Our ribonucleoprotein-based genome-editing system has a great advantage over conventional and in vivo CRISPR/Cas9 methods for genome editing in C. purpurea, which will greatly facilitate elucidation of the EAS biosynthetic pathway and other future basic and applied research on C. purpurea.