gene modification
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Author(s):  
Yanchun Wang ◽  
Shuli Sang ◽  
Xin Zhang ◽  
Haoxia Tao ◽  
Qing Guan ◽  
...  

A miniature CRISPR-Cas12f has been demonstrated to serve as an effective genome editing tool in gram negative bacteria as well as human cells. Here, we developed an alternative method to edit the genome of Bacillus anthracis based on the AsCas12f1 nuclease from Acidibacillus sulfuroxidans. When the htrA gene on the chromosome and the lef gene on the plasmid pXO1 were selected as targets, the CRISPR-AsCas12f1 system showed very high efficiency (100%). At the same time, a high efficiency was observed for large-fragment deletion. Our results also indicated that the length of the homologous arms of the donor DNA had a close relationship with the editing efficiency. Furthermore, a two-plasmid CRISPR-AsCas12f1 system was also constructed and combined with the endonuclease I-SceI for potential multi-gene modification. This represents a novel tool for mutant strain construction and gene function analyses in B. anthracis and other Bacillus cereus group bacteria.


2021 ◽  
Vol 7 (12) ◽  
pp. 1094
Author(s):  
Li Li ◽  
Na Xu ◽  
Fusheng Chen

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries and are currently used around the world via Asian catering. The MPs biosynthetic pathway has been well-illustrated; however, the functions of a few genes including mrpigH in the MPs gene cluster of M. ruber M7 are still unclear. In the current study, mrpigH was disrupted in Δmrlig4ΔmrpyrG, a highly efficient gene modification system, using mrpyrG as a selection marker, and ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG have been obtained. Subsequently, their morphologies, biomasses, MPs and citrinin (CIT) production were analyzed, respectively. These results have revealed that the deletion of mrpigH has significant effects on the morphology and growth of M. ruber M7. Moreover, compared with M. ruber M7, the yields of MPs and CIT were drastically increased and decreased in mrpigH mutants, respectively.


Author(s):  
Li Li ◽  
Na Xu ◽  
Fusheng Chen

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries and nowadays in the whole world via Asian catering. The MPs biosynthetic pathway has been well-illustrated, however, the functions of a few genes including mrpigH in the MPs gene cluster of M. ruber M7 are still unclear. In current study, mrpigH was disrupted in Δmrlig4ΔmrpyrG, a highly efficient gene modification system, using mrpyrG as a selection marker, and ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG have been obtained. Subsequently, their morphologies, biomasses, MPs and citrinin (CIT) production were analyzed, respectively. These results have revealed that the deletion of mrpigH has significant effects on the morphology and growth of M. ruber M7. Moreover, compared with M. ruber M7, the yields of MPs and CIT were drastically increased and decreased in mrpigH mutants, respectively.


2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi216-vi216
Author(s):  
Mandy Taisto ◽  
Barbara Tschida ◽  
Alexander Coutts ◽  
Jarryd Campbell ◽  
Stanislas Corbiere ◽  
...  

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor. Novel therapeutic development for GBM is desperately needed, as the standard of care universally fails to cure patients and the 5-year survival rate remains extremely low. GBM therapeutic development is hampered by the lack of relevant preclinical models for preclinical studies. To mitigate this problem, we have developed a genetic model of GBM in outbred, immune-proficient swine which have comparable brain size and anatomy to humans. We developed methods for introducing genome engineering tools to minipig brain in vivo by direct injection of gene delivery reagents to the lateral ventricle, altering major signaling pathways frequently changed in human GBM. Using this technique, we have delivered a combination of expression vectors for oncogenes and targeted nucleases to disrupt tumor suppressor genes commonly altered in human GBM. We have altered six major human GBM-associated signaling pathways and modeled molecular GBM subclasses. We have also engineered a secreted tumor reporter that can be used to monitor tumor size through a simple blood test. This somatic cell gene-modification platform we have developed in the minipig allows us to reproduce the genetic heterogeneity seen in GBM and understand the impact of the tumor microenvironment, immune system, and response to therapy. This minipig model of GBM Is being used to test the standard of care against novel therapies in preclinical studies, and biopsy, surgical, imaging, and radiation therapy techniques are being optimized in this mode to improve clinical trial success rates and patient outcomes. Funding for this study is provided by the National Institutes of Health though SBIR grant # 1R43CA235837-01A1.


Author(s):  
Dong-Joon Lee ◽  
Hyun-Yi Kim ◽  
Seung-Jun Lee ◽  
Han-Sung Jung

Over the past 40 years, studies on tooth regeneration have been conducted. These studies comprised two main flows: some focused on epithelial–mesenchymal interaction in the odontogenic region, whereas others focused on creating a supernumerary tooth in the non-odontogenic region. Recently, the scope of the research has moved from conventional gene modification and molecular therapy to genome and transcriptome sequencing analyses. However, these sequencing data have been produced only in the odontogenic region. We provide RNA-Seq data of not only the odontogenic region but also the non-odontogenic region, which loses tooth-forming capacity during development and remains a rudiment. Sequencing data were collected from mouse embryos at three different stages of tooth development. These data will expand our understanding of tooth development and will help in designing developmental and regenerative studies from a new perspective.


Children ◽  
2021 ◽  
Vol 8 (10) ◽  
pp. 862
Author(s):  
Rita P. Verma

Congenital anomalies (CA) are a large heterogeneous group of disorders of abnormal morphogenesis or biochemistry which present at birth and carry widely variable implications for morbidity and mortality. They are the leading cause of infant mortality in the USA, with an incidence of 3–4% of all births. CA are the fourth leading cause of neonatal mortality worldwide, with an estimated 295,000 deaths annually. The enormous variability in the clinical presentation in terms of severity, time of occurrence, course, complications, management, and outcomes makes the evaluation of CA complicated, highly specific, and individualized. The anomalies can impart tremendous physical, social, and emotional distress on the patient with massive emotional, social, financial, and medical implications for the family and society. The diagnosis may remain elusive despite rigorous, elaborate, and extensive investigations in many cases. While the enormous strides in genetic testing and gene modification therapy have an encouraging impact on the diagnosis and treatment, the risk assessment of recurrence in the family and population of CA remains obscure in most cases due to the lack of information and referable evidence.


2021 ◽  
Author(s):  
Junjie Zheng ◽  
Daiwei Chen ◽  
Cuiyu Zhang ◽  
Yuge Ji ◽  
Yanting Liu ◽  
...  

Abstract Background Gradually, more researches pay attention to the potential relationship between N6-methyladenosine (m6A) modification of RNA and immunity in tumor microenvironment (TME). Therefore, they developed unique scoring modes through detecting level of m6A in various tumors to learn immune phenotypes. Nevertheless, direct detection always is harmful even fatal to patients. Recent studies demonstrated that exosomes in body fluid would deliver RNA with m6A modification from tumors. Methods Through analyzing datasets from 1066 colon cancer samples, m6A-related exosome genes modification patterns which based on 59 m6A-related exosome genes was classified. Then we used MREGS to calculate modification patterns of each tumor with PCA algorithm. Results In accordance with 59 m6A-related exosome gene, we indicated that there were distinguishing characteristics of TME cell infiltration in three different m6A-related exosome gene modification patterns. Three immune cell infiltration profiles (immune-inflamed, immune-desert and immune-excluded) were pronouncedly relevant with the identified patterns in aspects of immune activation, EMT- and TGFβ-related pathways and activity of stroma in TME. We also discovered that tumor molecular subtypes, genetic aberrant expression and patients’ outcomes could be evaluated by identified modification patterns. Furthermore, patients in lower MREGS group, featured as prolonged lifespan and advantageous immune activation, was associated with high tumor mutation burden (TMB) and good prognosis with anti-PDL1 immune therapy. Conclusion Detecting the level of m6A methylation of exosomes in patients’ serum, MREGS could be utilized for assessing m6A-related exosome gene modification patterns of each patient and their own matching characteristics of TME cell infiltration, and next ascertain immunological classifications of tumors and assist clinicians to tailor optimal treatment for patients.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Guiping Zeng ◽  
Zhong Li ◽  
Zhi Zhao

AbstractThe fungus Wolfiporia cocos has wide-ranging and important medicinal value, and its dried sclerotia are used as a traditional Chinese medicine. Modern studies have shown that triterpenoid, the active ingredient of W. cocos, have a variety of pharmacological effects. The aim of our research was to determine the key genes related to triterpenoid biosynthesis, which may be useful for the genetic modification of cell-engineered bacteria for triterpenoid biosynthesis. In this study, two monospore strains, DZAC-WP-H-29 (high-yielding) and DZAC-WP-L-123 (low-yielding), were selected from the sexually propagated offspring of strain 5.78 of W. cocos, and the mycelia were cultured for 17, 34, and 51 days, respectively. Weighted gene co-expression network analysis (WGCNA) method was used to analyze transcriptional expressions. The results show that eight core genes (ACAT1-b, hgsA, mvd1, SQLE, erg6, TAT, erg26, and erg11) are associated with the triterpenoid synthesis pathway, and Pm20d2 and norA outside the pathway may be important genes that influence the biosynthesis and accumulation of W. cocos triterpenoid. The biosynthesis of W. cocos triterpenoid is closely related to the expression of sterol metabolic pathway genes. The role of these genes in triterpenoid synthesis complements our knowledge on the biosynthesis and accumulation of W. cocos triterpenoid, and also provides a reference for the target gene modification of engineered bacteria for the fermentation production of triterpenoid.


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