scholarly journals Fast, Efficient, and Precise Gene Editing in the MossPhyscomitrella patens

2019 ◽  
Author(s):  
Peishan Yi ◽  
Gohta Goshima
Keyword(s):  
Gene Editing ◽  
High Efficiency ◽  
Physcomitrella Patens ◽  
Gene Knockout ◽  
Model Organism ◽  
Plant Evolution ◽  
High Rate ◽  
Ideal System ◽  
Redundant Genes ◽  
One Step

AbstractRecent years, the bryophyte mossPhyscomitrella patenshas become an emerging model organism for studying conserved signaling pathways and developmental processes during plant evolution. Its short life cycle, ease of cultivation, and high rate of homologous recombination have made it an ideal system for genetic analysis. However, the presence of highly redundant genes and the difficulty of isolating hypomorphic mutants have limited its broader use. Here we developed a simple, fast, and efficient method to generate customized mutants inP. patens.We show that transient cotransformation of CRISPR/Cas9 and oligonucleotide templates enables microindel knock-in with high efficiency and accuracy. Using this method, we generated strains carrying various types of mutations, including amino acid substitution, out-of-frame deletion/insertion, splice site alteration, and small tag integration. We also demonstrate that multiplex gene editing can be efficiently achieved to generate putative null and hypomorphic mutants of redundant genes in one step. Thus our method will not only simplify multiple-gene knockout, but also allows the generation of hypomorphic mutants of genes of interest, especially those that are essential for viability.

scholarly journals One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs

Cell Research ◽  
2017 ◽  
Vol 27 (7) ◽  
pp. 933-945 ◽  
Author(s):  
Erwei Zuo ◽  
Yi-Jun Cai ◽  
Kui Li ◽  
Yu Wei ◽  
Bang-An Wang ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Gene Editing ◽  
Gene Knockout ◽  
Gene Knockout Mice ◽  
One Step ◽  
Step Generation

scholarly journals The MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and Overexpression CRISPR-Cas9 Toolkit

2021 ◽  
Author(s):  
Bjorn DM Bean ◽  
Malcolm Whiteway ◽  
Vincent JJ Martin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Efficiency ◽  
Model Organism ◽  
Basic Research ◽  
Expression Vectors ◽  
Selectable Markers ◽  
Guide Rna ◽  
One Step ◽  
Selection For ◽  
The One

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful CRISPR-Cas9-based genome editing tools we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit with three components: (i) a set of optimized S. pyogenes Cas9-guide RNA (gRNA) expression vectors with five selectable markers and the option to either pre-clone or co-transform the gRNAs; (ii) vectors for the one-step construction of integration cassettes expressing an untagged or GFP/RFP/HA-tagged gene of interest at one of three levels, supporting localization and overexpression studies; and (iii) integration cassettes containing moderately expressed GFP- or RFP-tagged compartmental markers for colocalization experiments. These components allow rapid, high efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle to simplify yeast research

scholarly journals Targeted Gene Knockouts by Protoplast Transformation in the Moss Physcomitrella patens

2021 ◽  
Vol 3 ◽  
Author(s):  
Lei Zhu
Keyword(s):  
Gene Targeting ◽  
Physcomitrella Patens ◽  
Gene Knockout ◽  
Model Organism ◽  
Gene Replacement ◽  
Protoplast Transformation ◽  
End Joining ◽  
Gene Functions ◽  
Technical Challenges

Targeted gene knockout is particularly useful for analyzing gene functions in plant growth, signaling, and development. By transforming knockout cassettes consisting of homologous sequences of the target gene into protoplasts, the classical gene targeting method aims to obtain targeted gene replacement, allowing for the characterization of gene functions in vivo. The moss Physcomitrella patens is a known model organism for a high frequency of homologous recombination and thus harbors a remarkable rate of gene targeting. Other moss features, including easy to culture, dominant haploidy phase, and sequenced genome, make gene targeting prevalent in Physcomitrella patens. However, even gene targeting was powerful to generate knockouts, researchers using this method still experienced technical challenges. For example, obtaining a good number of targeted knockouts after protoplast transformation and regeneration disturbed the users. Off-target mutations such as illegitimate random integration mediated by nonhomologous end joining and targeted insertion wherein one junction on-target but the other end off-target is commonly present in the knockouts. Protoplast fusion during transformation and regeneration was also a problem. This review will discuss the advantages and technical challenges of gene targeting. Recently, CRISPR-Cas9 is a revolutionary technology and becoming a hot topic in plant gene editing. In the second part of this review, CRISPR-Cas9 technology will be focused on and compared to gene targeting regarding the practical use in Physcomitrella patens. This review presents an updated perspective of the gene targeting and CRISPR-Cas9 techniques to plant biologists who may consider studying gene functions in the model organism Physcomitrella patens.

One-step Separation and Purification of Four Phenolic Acids from Stenoloma chusanum (L.) Ching by Medium-pressure Liquid Chromatography and High-speed Counter-current Chromatography

2019 ◽  
Vol 9 (2) ◽  
pp. 138-143
Author(s):  
Tianyun Li ◽  
Xiling Dai ◽  
Yichen Li ◽  
Guozheng Huang ◽  
Jianguo Cao
Keyword(s):  
Natural Products ◽  
Liquid Chromatography ◽  
Phenolic Acids ◽  
High Speed ◽  
High Efficiency ◽  
Total Phenolic ◽  
Medium Pressure ◽  
Medium Pressure Liquid Chromatography ◽  
One Step ◽  
Counter Current

Background:Stenoloma chusanum (L.) Ching is a Chinese traditional medicinal fern with high total flavonoid and total phenolic content. Traditionally, phenolic compounds were separated by using column chromatography, which is relatively inefficient. </P><P> Objective: This study aims to use an efficient method to separate natural products from S. chusanum by Medium-Pressure Liquid Chromatography (MPLC) and High-Speed Counter-Current Chromatography (HSCCC).Methods:In the present research, firstly, a sample (2.5 g) from the dichloromethane extract of S. chusanum was separated by MPLC. Next, fraction P5 was purified by HSCCC with a two-phase solvent system composed of hexane-ethyl acetate-methanol-water (HEMWat) at a volume ratio of 2:4:1:4 (v/v/v/v). </P><P> Result: Four phenolic acids were obtained and their structures were identified by means of NMR and ESI-mass analysis. They were identified as: 1) protocatechuic acid (34 mg, purity 90.1%), 2) syringic acid (66 mg, purity 99.0%), 3) p-hydroxybenzoic acid (5 mg, purity 91.2%) and 4) vanillic acid (6 mg, purity 99.3%).Conclusion:The combination of MPLC and HSCCC is a high-efficiency separation method for natural products. This is the first report with regard to the separation of four phenolic acids in one step by MPLC and HSCCC from S. chusanum (L.) Ching.

Well-designed honeycomb Co3O4@CdS photocatalyst derived from cobalt foam for high-efficiency visible-light H2 evolution

2021 ◽  
Author(s):  
Chao Zhang ◽  
Baoquan Liu ◽  
Weiping Li ◽  
Xiangxue Liu ◽  
Ke Wang ◽  
...  
Keyword(s):  
Visible Light ◽  
Water Splitting ◽  
High Efficiency ◽  
H2 Evolution ◽  
One Step

Well-designed honeycomb Co3O4@CdS (H-Co3O4@CdS) was fabricated via a one-step strategy for efficient water splitting. During the decoration of CdS, honeycomb Co3O4 (H-Co3O4) with macropore was formed simultaneously. H-Co3O4 could enhance...

scholarly journals Ultrafast presodiation of graphene anodes for high‐efficiency and high‐rate s odium‐ion storage

InfoMat ◽  
2021 ◽  
Author(s):  
Ganyu Zheng ◽  
Qiaowei Lin ◽  
Jiabin Ma ◽  
Jun Zhang ◽  
Yan‐Bing He ◽  
...  
Keyword(s):  
High Efficiency ◽  
High Rate ◽  
Ion Storage

scholarly journals Molecular Evolution of Phototransduction Pathway Genes in Nocturnal and Diurnal Fireflies (Coleoptera: Lampyridae)

Insects ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 561
Author(s):  
Gavin J. Martin ◽  
Sarah E. Lower ◽  
Anton Suvorov ◽  
Seth M. Bybee
Keyword(s):  
Positive Selection ◽  
Transient Receptor Potential ◽  
Strong Support ◽  
Model Organism ◽  
Receptor Potential ◽  
Guanine Nucleotide Binding Protein ◽  
Weak Support ◽  
Niche Adaptation ◽  
Ideal System ◽  
Phototransduction Pathway

Most organisms are dependent on sensory cues from their environment for survival and reproduction. Fireflies (Coleoptera: Lampyridae) represent an ideal system for studying sensory niche adaptation due to many species relying on bioluminescent communication; as well as a diversity of ecologies. Here; using transcriptomics; we examine the phototransduction pathway in this non-model organism; and provide some of the first evidence for positive selection in the phototransduction pathway beyond opsins in beetles. Evidence for gene duplications within Lampyridae are found in inactivation no afterpotential C and inactivation no afterpotential D. We also find strong support for positive selection in arrestin-2; inactivation no afterpotential D; and transient receptor potential-like; with weak support for positive selection in guanine nucleotide-binding protein G(q) subunit alpha and neither inactivation nor afterpotential C. Taken with other recent work in flies; butterflies; and moths; this represents an exciting new avenue of study as we seek to further understand diversification and constraint on the phototransduction pathway in light of organism ecology.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

A Powerful One‐Step Puffing Carbonization Method for Construction of Versatile Carbon Composites with High‐Efficiency Energy Storage

2021 ◽  
pp. 2102796
Author(s):  
Shenghui Shen ◽  
Lei Huang ◽  
Xili Tong ◽  
Rongfan Zhou ◽  
Yu Zhong ◽  
...  
Keyword(s):  
Energy Storage ◽  
High Efficiency ◽  
Carbon Composites ◽  
One Step
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