Monday, August 17, 2020

Multi Crispr Knockout Bacteria

 Scientists have developed a continuous knockout cell line technology based on the CRISPR-Cas9 system, including single and multiple (up to three) target gene insertions and knockouts for the purpose of bacterial modification. The CRISPR/Cas system is a kind of prokaryotic immunity. The resistance of the system to foreign genetic elements (such as those found in plasmids and phages) can provide some form of acquired immunity. RNA with spacer sequence can help Cas protein recognize and cleave foreign DNA. Other Cas proteins guided by RNA can cleave foreign RNA. CRISPR is found in approximately 40% of bacterial genome sequences and 90% of archaeal sequences.

Multi-gene editing in the E. coli genome through the CRISPR-Cas9 system

The construction of industrially useful microorganisms requires effective genome-scale editing tools. The researchers describe a targeted, continuous multi-gene editing strategy that uses the Streptococcus pyogenes type II CRISPR-Cas9 system to be applied to the E. coli genome to achieve a variety of precise genome modifications, including gene deletion and insertion, with efficiency The maximum is 100%, and it can perform multi-gene editing on three targets at the same time. The system also proved that it successfully achieved targeted chromosome deletion in another Enterobacteriaceae-Tatumella citrea, with an efficiency of up to 100%.

Multi-stage knockout of Escherichia coli using CRISPR/Cas9

With the recent use of CRISPR/Cas9 technology as a standard tool for genome editing, laboratories around the world have experienced one of the largest molecular biology advances since PCR. The main advantage of this method is its simplicity and versatility for any category. Of particular interest is the widely studied Gram-negative bacterium Escherichia coli, because it is considered a major force for research and industrial applications. The researchers proposed a simple, reliable and effective scheme using the CRISPR/Cas9 system combined with the lambda Red machine for gene knockout in E. coli. In our procedure, it is crucial to use double-stranded donor DNA and a solidification strategy to remove the RNA-encoding guide plasmid, which allows the initiation of new mutations in only two working days. Our protocol allows multiple stepwise knockout strains with high mutagenesis efficiency to be suitable for high-throughput methods.

Multi-functional genome-wide CRISPR system for high throughput genotype–phenotype mapping

Genome-scale engineering is an indispensable tool to understand genome functions due to our limited knowledge of cellular networks. Unfortunately, most existing methods for genome-wide genotype–phenotype mapping are limited to a single mode of genomic alteration, i.e. overexpression, repression, or deletion. Researcher report a multi-functional genome-wide CRISPR (MAGIC) system to precisely control the expression level of defined genes to desired levels throughout the whole genome. By combining the tri-functional CRISPR system and array-synthesized oligo pools, MAGIC is used to create, to the best of our knowledge, one of the most comprehensive and diversified genomic libraries in yeast ever reported. The power of MAGIC is demonstrated by the identification of previously uncharacterized genetic determinants of complex phenotypes, particularly those having synergistic interactions when perturbed to different expression levels. MAGIC represents a powerful synthetic biology tool to investigate fundamental biological questions as well as engineer complex phenotypes for biotechnological applications.

Ubigene developed CRISPR-B™ which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi

Reference

Endo M, Mikami M, Toki S. Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant Cell Physiol. 2015;56(1):41-47. doi:10.1093/pcp/pcu154

König, Enrico & Zerbini, Francesca & Zanella, Ilaria & Fraccascia, Davide & Grandi, Guido. (2018). Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli. BIO-PROTOCOL. 7. 10.21769/BioProtoc.2688.

Jiazhang Lian, Carl Schultz, Mingfeng Cao, Mohammad HamediRad,Huimin Zhao.Multi-functional genome-wide CRISPR system for high throughput genotypephenotype mapping.Nature Communications .2019.

The efficiency of gene knock-out and cleavage can not only give people the ability to generate protein radical profiles and establish regulatory records, but also has many advantages, making it a particularly attractive recombinant protein expression system. First, it is carboxylated on glutamic acid and sulfated on tyrosine. Second, the operation is simple, and the recombinant protein can be quickly produced through transient gene expression. Third, it can be used for stable recombinant protein production. Some researchers used gene cell knockout and cutting efficiency systems to generate gene-edited cell lines, targeted sequencing of GLUL genomic loci, produced stable EPO cell lines, and discovered the mechanism of stable expression of recombinant erythropoietin in humans .

According to customer needs, Yuanjing Biotechnology designs a stable gene transfer knockout program based on the target gene.
Scheme 1: Small-segment gene knockout program, gRNA is set in the introns at both ends of exon 2, and the number of bases encoded by the knockout exon is not 3 times, and the knockout can cause frameshift.
Scheme 2: Frameshift gene knockout scheme, gRNA is set on the exon, the number of missing bases is not 3 times, and frameshift mutation can occur after knockout.
Scheme 3: Large-segment gene knockout scheme, knock out the coding sequence of the entire gene to achieve the effect of large-segment knockout.

Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.

We developed CRISPR-U™ and CRISPR-B™(based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, CRISPR-U™ and CRISPR-B™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. 

Genome Editing Platform
——Focusing on the Application of CRISPR-U™ and CRISPR-B™ Gene Editing Technology
1. Provides various types of gene-editing vectors for different species.
2. Provides different virus packaging services, including lentiviruses, adenoviruses and adeno-associated viruses.3. Provides high-quality services for gene knockout, point mutation and knockin cell lines

Cell Biology Platform
——Focusing on primary cell
1. Provides over 400 types of primary cells.
2. Provides culture strategies and related products for different cell types.3. Provides cell biology-related services such as cell isolation, extraction and validation.

Tuesday, August 11, 2020

Eliminate Escherichia coli

 Escherichia coli (also known as Escherichia coli) is a Gram-negative, facultative anaerobic, rod-shaped coliform of Escherichia coli, usually found in the lower intestine (endothermic) of warm-blooded organisms. Most E. coli strains are harmless, but certain serotypes can cause severe food poisoning in the host, and sometimes product recalls due to food contamination. Harmless bacterial strains are part of the normal intestinal flora and can have a symbiotic relationship by producing vitamin K2 and preventing pathogenic bacteria from colonizing in the intestine, thereby benefiting the host. Escherichia coli is excreted in feces. The bacteria grew in large numbers in fresh feces for 3 days under aerobic conditions, but then the number slowly decreased. Due to the long history of laboratory culture and easy operation, Knockout Cell Lines E. coli plays an important role in modern bioengineering and industrial microbiology. [82] The work of Stanley Norman Cohen and Herbert Boyer in E. coli, using plasmids and restriction enzymes to produce recombinant DNA, became the basis of biotechnology.

Metabolic flux analysis of pykF knockout Escherichia coli based on 13C labeling experiment and measurement of enzyme activity and intracellular metabolite concentration

Metabolic flux analysis based on 13C labeling experiment was carried out, and then 2D NMR and GC-MS were used to measure intracellular isotope distribution to study the effect of pyruvate kinase (pyk) gene knockout on the metabolism of continuously cultured E. coli. In addition, in batch culture and continuous culture, the activity of 16 enzymes and the concentration of 5 intracellular metabolites were measured as a function of time. It was found that the flux through phosphoenolpyruvate carboxylase and malate was up-regulated in the pykF-mutant compared to the wild-type, and the formation of acetate was significantly reduced in the mutant. In addition, in the mutant, the flux through the phosphofructokinase pathway was reduced, while the flux through the oxidized pentose phosphate (PP) pathway was increased. This is evidenced by the corresponding enzymatic activity and increased concentrations of phosphoenolpyruvate, 6-phosphate glucose and 6-phosphate gluconate. It was also found that for continuous culture, the enzyme activity of oxidizing PP and Entner-Doudoroff. The dilution of the pykF-mutant increases with the increase of the pathway. In order to clarify the metabolism quantitatively, it is important to find that it is important to integrate information about the intracellular metabolic flux distribution, enzyme activity and intracellular metabolite concentration.

Using CRISPR-Cas9 to reconstruct the TCA cycle involving I-isoleucine dioxygenase to hydroxylate I-isoleucine in E. coli

L-isoleucine dioxygenase (IDO) is an iron (II)/α-ketoglutarate (α-KG) dependent dioxygenase, which can convert l-isoleucine (l-Ile) Specifically converted to (2S, 3R, 4S)-4-hydroxyisoleucine (4-HIL). 4-HIL is an important drug for the treatment and prevention of type 1 and type 2 diabetes, but the current method has a low yield. In this study, the CRISPR-Cas9 gene editing system was used to knock out the sucAB and aceAK genes in the TCA cycle pathway of E. coli. For single gene knockout, the entire process takes about 7 days. However, the time for each round of genetic modification for multiple rounds of editing was reduced by 2 days. Using the genome-edited recombinant strain Escherichia coli BL21(DE3)ΔsucABΔaceAK/ pET-28a(+)-ido(2Δ-ido), compared with E., the biotransformation rate of 4-HIL by L-Ile increased by about 15% . Escherichia coli BL21(DE3)/pET-28a(+)-ido [BL21(DE3)-ido] strain. The CRISPR-Cas9 editing strategy has the potential to modify multiple genes more quickly and optimize strains for industrial production.

CRISPR-Cas9 knocking out qseB induces asynchrony between E. coli motility and biofilm formation

Generally, cell motility and biofilm formation are tightly regulated. The QseBC two-component system (TCS) acts as a bridge for bacterial signal transmission, where the QseB protein acts as a response regulator of bacterial motility, biofilm formation and virulence. In general, the mechanism that controls the interaction between QseBC and its functions has been studied, but it is not clear how QseB regulates bacterial motility and biofilm formation. In this study, the E. coli MG1655ΔqseB strain (strain ΔqseB) was constructed using the CRISPR-Cas9 system, and the influence of the qseB gene on wild-type (WT) motility and biofilm formation was determined. The results of vigor determination showed that the ΔqseB strain had higher (p<0.05) vigor than the WT strain. However, there was no difference in biofilm formation between ΔqseB and WT strains. real-

Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.

We developed CRISPR-U™ and CRISPR-B™(based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, CRISPR-U™ and CRISPR-B™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. 



Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.

We developed CRISPR-U™ and CRISPR-B™(based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, CRISPR-U™ and CRISPR-B™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. 

Genome Editing Platform
——Focusing on the Application of CRISPR-U™ and CRISPR-B™ Gene Editing Technology
1. Provides various types of gene-editing vectors for different species.
2. Provides different virus packaging services, including lentiviruses, adenoviruses and adeno-associated viruses.3. Provides high-quality services for gene knockout, point mutation and knockin cell lines. 

Cell Biology Platform
——Focusing on primary cell
1. Provides over 400 types of primary cells.
2. Provides culture strategies and related products for different cell types.3. Provides cell biology-related services such as cell isolation, extraction and validation.

Friday, August 7, 2020

Multi Crispr Knockout Bacteria

Scientists have developed a continuous knockout cell line technology based on the CRISPR-Cas9 system, including single and multiple (up to three) target gene insertions and knockouts for the purpose of bacterial modification. The CRISPR/Cas system is a kind of prokaryotic immunity. The resistance of the system to foreign genetic elements (such as those found in plasmids and phages) can provide some form of acquired immunity. RNA with spacer sequence can help Cas protein recognize and cleave foreign DNA. Other Cas proteins guided by RNA can cleave foreign RNA. CRISPR is found in approximately 40% of bacterial genome sequences and 90% of archaeal sequences.

Multi-gene editing in the E. coli genome through the CRISPR-Cas9 system

The construction of industrially useful microorganisms requires effective genome-scale editing tools. The researchers describe a targeted, continuous multi-gene editing strategy that uses the Streptococcus pyogenes type II CRISPR-Cas9 system to be applied to the E. coli genome to achieve a variety of precise genome modifications, including gene deletion and insertion, with efficiency The maximum is 100%, and it can perform multi-gene editing on three targets at the same time. The system also proved that it successfully achieved targeted chromosome deletion in another Enterobacteriaceae-Tatumella citrea, with an efficiency of up to 100%.

Multi-stage knockout of Escherichia coli using CRISPR/Cas9

With the recent use of CRISPR/Cas9 technology as a standard tool for genome editing, laboratories around the world have experienced one of the largest molecular biology advances since PCR. The main advantage of this method is its simplicity and versatility for any category. Of particular interest is the widely studied Gram-negative bacterium Escherichia coli, because it is considered a major force for research and industrial applications. The researchers proposed a simple, reliable and effective scheme using the CRISPR/Cas9 system combined with the lambda Red machine for gene knockout in E. coli. In our procedure, it is crucial to use double-stranded donor DNA and a solidification strategy to remove the RNA-encoding guide plasmid, which allows the initiation of new mutations in only two working days. Our protocol allows multiple stepwise knockout strains with high mutagenesis efficiency to be suitable for high-throughput methods.

Multi-functional genome-wide CRISPR system for high throughput genotype–phenotype mapping

Genome-scale engineering is an indispensable tool to understand genome functions due to our limited knowledge of cellular networks. Unfortunately, most existing methods for genome-wide genotype–phenotype mapping are limited to a single mode of genomic alteration, i.e. overexpression, repression, or deletion. Researcher report a multi-functional genome-wide CRISPR (MAGIC) system to precisely control the expression level of defined genes to desired levels throughout the whole genome. By combining the tri-functional CRISPR system and array-synthesized oligo pools, MAGIC is used to create, to the best of our knowledge, one of the most comprehensive and diversified genomic libraries in yeast ever reported. The power of MAGIC is demonstrated by the identification of previously uncharacterized genetic determinants of complex phenotypes, particularly those having synergistic interactions when perturbed to different expression levels. MAGIC represents a powerful synthetic biology tool to investigate fundamental biological questions as well as engineer complex phenotypes for biotechnological applications.

Ubigene developed CRISPR-B™ which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi

Reference

Endo M, Mikami M, Toki S. Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant Cell Physiol. 2015;56(1):41-47. doi:10.1093/pcp/pcu154

König, Enrico & Zerbini, Francesca & Zanella, Ilaria & Fraccascia, Davide & Grandi, Guido. (2018). Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli. BIO-PROTOCOL. 7. 10.21769/BioProtoc.2688.

Jiazhang Lian, Carl Schultz, Mingfeng Cao, Mohammad HamediRad,Huimin Zhao.Multi-functional genome-wide CRISPR system for high throughput genotypephenotype mapping.Nature Communications .2019.

The efficiency of gene knock-out and cleavage can not only give people the ability to generate protein radical profiles and establish regulatory records, but also has many advantages, making it a particularly attractive recombinant protein expression system. First, it is carboxylated on glutamic acid and sulfated on tyrosine. Second, the operation is simple, and the recombinant protein can be quickly produced through transient gene expression. Third, it can be used for stable recombinant protein production. Some researchers used gene cell knockout and cutting efficiency systems to generate gene-edited cell lines, targeted sequencing of GLUL genomic loci, produced stable EPO cell lines, and discovered the mechanism of stable expression of recombinant erythropoietin in humans .

According to customer needs, Yuanjing Biotechnology designs a stable gene transfer knockout program based on the target gene.
Scheme 1: Small-segment gene knockout program, gRNA is set in the introns at both ends of exon 2, and the number of bases encoded by the knockout exon is not 3 times, and the knockout can cause frameshift.
Scheme 2: Frameshift gene knockout scheme, gRNA is set on the exon, the number of missing bases is not 3 times, and frameshift mutation can occur after knockout.
Scheme 3: Large-segment gene knockout scheme, knock out the coding sequence of the entire gene to achieve the effect of large-segment knockout.

Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.

We developed CRISPR-U™ and CRISPR-B™(based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, CRISPR-U™ and CRISPR-B™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. 

Genome Editing Platform
——Focusing on the Application of CRISPR-U™ and CRISPR-B™ Gene Editing Technology
1. Provides various types of gene-editing vectors for different species.
2. Provides different virus packaging services, including lentiviruses, adenoviruses and adeno-associated viruses.3. Provides high-quality services for gene knockout, point mutation and knockin cell lines

Cell Biology Platform
——Focusing on primary cell
1. Provides over 400 types of primary cells.
2. Provides culture strategies and related products for different cell types.3. Provides cell biology-related services such as cell isolation, extraction and validation.

Wednesday, August 5, 2020

KYSE-150 CRISPR cell line | 100% guarantee | High-efficiency

KYSE-150 cells are poorly differentiated esophageal adenocarcinoma cells that were isolated from the neck esophagus of a 49-year-old Japanese female patient who had received radiotherapy. When researchers discovered this cell line, the patient's cancer tissue had invaded the adjacent tissues. The KYSE-150 cell line is an epithelial cell with an adherent monolayer. It has been reported that cells carry increased oncogenes c-erb-B (8 times) and cyclin D1 (4 times) and that cells can form tumors in nude mice, so KYSE-150 cell line is often used in cancer research involving gene editings, such as CRISPR gene knockout/knock-in and point mutation.

 Applications:

1. EZH2 knockout in KYSE-150 cells altered PSMA3-AS1-induced proliferation and migration in esophageal cancer cells

PSMA3-AS1 expression is up-regulated and positively correlated with tumor size and metastasis in esophageal cancer patients. To further explore the biological roles of PSMA3-AS1 in esophageal cancer cells, researchers established stable CRISPR PSMA3-AS1-overexpressing cell lines via lentiviral infection in KYSE150 and KYSE450 cell lines (which have low PSMA3-AS1 expression) and validated the up-regulation of PSMA3-AS1 by RT-qPCR.

CRISPR/Cas9 gene-editing of EZH2 was successfully performed in KYSE150 and KYSE450cells as confirmed by a significant reduction in EZH2 protein expression. CCK-8 and colony formation assays showed that PSMA3-AS1 overexpression did not affect the proliferation of esophageal cancer cells with CRISPR EZH2 knocked out (Figure 6B and 6C). According to wound healing and transwell migration assays, wounding healing and cell migration were not increased in ESCC EZH2-knock out KYSE-150 cells compared to negative control cells.

Clinical-pathological characteristics illustrated that increased expression of PSMA3-AS1 was positively associated with distant metastasis, larger tumor sizes, and a worse prognosis for ESCC patients.


 2. CRISPR Over-expressed KYSE-150 cells increase chemo-resistance in ESCC

Whole-genome clustered regularly interspaced short palindromic repeats/CRISPR associated (CRISPR/Cas)-based a lentiviral library is a powerful tool for genome-scale gain-of-function or loss-of-function screening. This system has been proved to be highly effective in identifying drug-resistant genes in vitro. Shalem, Kurata, and Joung have screened out essential genes for drug resistance in melanoma and AML using the CRISPR knockout library. Some researchers attempted to combine the CRISPR library screening strategy with RNA sequencing technology in KYSE-150 Cells to explore the critical genes and potential mechanism for chemo-resistance in ESCC.

To increase the chance of identifying essential genes involved in PTX resistance, an integrated analysis was performed to combine EN-genes in genome-scale CRISPR screening and differentially expressed genes (DE-genes) in KYSE-150 cell line.

Researchers evaluated the potential of CDKN1A, ELAVL2, and TSPAN4 to promote chemo-resistance in ESCC cells KYSE-150. The results showed that overexpression of CDKN1A, ELAVL2, or TSPAN4 could significantly increase the resistance to PTX in both KYSE-180 and KYSE-150 cells. Moreover, overexpressed CDKN1A, ELAVL2, or TSPAN4 could also contribute to DDP-resistance in KYSE-150 cells.


 3. CRISPR/Cas9 mediated knockout of DEPTOR in KYSE-510 cells significantly promoted cellular proliferation, migration, and invasion

Researchers found that the expression of DEPTOR negatively regulates the tumorigenic activities of ESCC cell lines. Furthermore, ectopic DEPTOR expression caused significant suppression of the cellular proliferation, migration, and invasion of KYSE150 cells, which has the lowest expression level of DEPTOR. Meanwhile, CRISPR/Cas9 mediated knockout of DEPTOR in KYSE-510 cells significantly promoted cellular proliferation, migration, and invasion. Besides, in vivo assays further revealed that tumor growth was significantly inhibited in xenografts with ectopic DEPTOR expression as compared to untreated KYSE150 cells, and was markedly enhanced in DEPTOR knockout KYSE-510 cells.

In this study, scientists generated stable cell lines that either overexpressing DEPTOR or genetic ablation of endogenous expression of DEPTOR. Since KYSE-150 expresses the lowest endogenous level of DEPTOR among the three cell lines, they stably overexpressed DEPTOR in KYSE-150 cells (pcDNA3.1-DEPTOR). For the same consideration, KYSE-510 cells that express the highest level of DEPTOR were treated with CRISPR/Cas9 system to knockout of DEPTOR (CRISPR-DEPTOR). After the generation of cell lines, pcDNA3.1-DEPTOR displayed a reduced cell proliferation rate as compared to that of KYSE-150 parental cells and empty vector-transfected cells, while CRISPR-DEPTOR cells proliferated significantly faster than control KYSE-510 cells. Furthermore, pcDNA3.1-DEPTOR cells also showed reduced migration. Thus, these results suggested DEPTOR indeed regulates cell proliferation, migration, and invasion in ESCC cells.

Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.

We developed CRISPR-U™ and CRISPR-B™(based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, CRISPR-U™ and CRISPR-B™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. 


According to customer needs, Yuanjing Biotechnology designs a stable gene transfer knockout program based on the target gene.

Scheme 1: Small-segment gene knockout scheme, gRNA is set in the introns at both ends of exon 2, and the number of coding bases of the knockout exon is not 3 times, and the knockout can cause frame shift.

Scheme 2: Frameshift gene knockout scheme, gRNA is set on the exon, the number of missing bases is not 3 times, and frameshift mutation can occur after knockout.
Scheme 3: Large-segment gene knockout scheme, knock out the coding sequence of the entire gene to achieve the effect of large-segment knockout.

Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.

We developed CRISPR-U™ and CRISPR-B™(based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, CRISPR-U™ and CRISPR-B™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. 


References:

1. Nødvig CS, Hoof JB, Kogle ME, et al. Efficient oligonucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. Fungal Genet Biol. 2018;115:78-89

2.  Nødvig CS, Nielsen JB, Kogle ME, Mortensen, UH. A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLoS One. 2015;10(7):e0133085. Published 2015 Jul 15.

3. Vanegas, K.G., Jarczynska, Z.D., Strucko, T. et al. Cpf1 enables fast and efficient genome editing in Aspergilli. Fungal Biol Biotechnol 6, 6 (2019).

4. Nayak T, Szewczyk E, Oakley CE, et al. A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics. 2006;172(3):1557-1566. 


Genome Editing Platform

——Focusing on the Application of CRISPR-U™ and CRISPR-B™ Gene Editing Technology
1. Provides various types of gene-editing vectors for different species.
2. Provides different virus packaging services, including lentiviruses, adenoviruses and adeno-associated viruses.3. Provides high-quality services for gene knockout, point mutation and knockin cell lines

Cell Biology Platform
——Focusing on primary cell
1. Provides over 400 types of primary cells.

2. Provides culture strategies and related products for different cell types.3. Provides cell biology-related services such as cell isolation, extraction and validation.

CRISPR Pseudomonas Aeruginosa,Gene Editing Service

Pseudomonas aeruginosa is a common encapsulated, Gram-negative, rod-shaped bacterium that can cause disease in plants and animals, including humans. A species of considerable medical importance, P. aeruginosa is a multidrug resistant pathogen recognized for its ubiquity, its intrinsically advanced antibiotic resistance mechanisms, and its association with serious illnesses – hospital-acquired infections such as ventilator-associated pneumonia and various sepsis syndromes. This type of bacteria is found commonly in the environment, like in soil and in water. P. Aeruginosa is a gram-negative, aerobic rod bacterium of the Pseudomonadaceae family (a member of the Gammaproteobacteria). These years, P. aeruginosa are getting more popular in the field of CRISPR editing, which involves knockout cell lines, gene knock-in and point-mutation in the bacteria.


Applying CRISPR in P. aeruginosa Involving Insertion and Knockout of a Tag Gene

Pseudomonas aeruginosa is both a prototype multi-drug resistance (MDR) pathogen and a model species for CRISPR-Cas research. In a clinical setting that includes I-F CRISPR, this technology can be easily applied to the other two isolates of Pseudomonas aeruginosa. A two-step In-Del strategy was further developed, which involved inserting tags near the desired editing site, and then performing gene knockout to edit genomic sites lacking a valid PAM (protospacer adjacent motif) or essential genes. Among the three resistance mutations that enhance the resistance of fluoroquinolones, the gyrA mutation caused greater resistance compared with the efflux of MexAB-OprM or MexEF-OprN. These results promote the understanding of the development of MDR of clinical Pseudomonas aeruginosa strains and demonstrate the great potential of the natural CRISPR system in AMR research. Although there are complete genetic manipulation tools in various model strains, due to the diversity of DNA homeostasis and cytotoxicity of these strains, their application in "non-model" strains in medical, environmental and industrial senses is often affected. Hinder. Cloning of CRISPR-Cas9/Cpf1 system. The use of the natural CRISPR-Cas system with built-in genome targeting activity and widely distributed in prokaryotes provides a promising and effective method for solving these obstacles. The successful development of the first I-F CRISPR-mediated genome editing technology and its subsequent expansion to other clinical and environmental Pseudomonas aeruginosa isolates. Knockout Bacteria opens up a new approach to pathogen resistance functional genomics.


Silencing and Point Mutations in P. aeruginosa Helps Research in Bacterial Physiology, Drug Target Exploration, and Metabolic Engineering

Pseudomonas species exhibit significant biomedical, ecological, and industrial importance. Despite the extensive research and wide applications, genetic manipulation in Pseudomonas species, in particular in the major human pathogen Pseudomonas aeruginosa, remains a laborious endeavor. It is reported that a genome-editing method pCasPA/pACRISPR was developed by harnessing the CRISPR/Cas9 and the phage λ-Red recombination systems. The method allows for efficient and scarless genetic manipulation in P. aeruginosa. By engineering the fusion of the cytidine deaminase APOBEC1 and the Cas9 nickase, a base editing system pnCasPA-BEC was developed, which enables highly efficient gene inactivation and point mutations in a variety of Pseudomonas species, such as P. aeruginosa. Application of the two genome editing methods will dramatically accelerate a wide variety of investigations, such as bacterial physiology study, drug target exploration, and metabolic engineering.


The Rapid Construction of Gene Knockouts in P. aeruginosa with Base-pair Precision

Pseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune-compromised patients. As such P. aeruginosa is one of the most well-studied organisms in terms of its genetics. However, the construction of gene KOs and replacements in Pseudomonas aeruginosa is relatively time-consuming, requiring multiple steps including suicide vector construction, conjugation, inactivation with the insertion of antibiotic resistance cassettes and allelic exchange. Even employing Gateway recombineering techniques with direct transformation requires a minimum of two weeks. Hence, a rapid streamlined method was developed to create clean KO mutants in P. aeruginosa through direct transformation, eliminating the need for the creation of Gateway-compatible suicide vectors. In this method, upstream and downstream sequences of the gene/locus to be deleted are amplified by polymerase chain reaction (PCR) and seamlessly fused with the linearized pEX18Tc sacB suicide plasmid by Gibson assembly. The resulting knockout plasmid is transformed into P. aeruginosa by an electroporation method optimized in this study. The plasmid is then integrated into the chromosome by homologous recombination, and knockout mutants are identified via sacB mediated sucrose counter-selection. The current method was employed to generate clean gene knockouts of the heme assimilation system anti-σ factor, hasS, and the virulence regulator involving ECF system anti-σ and σ factors vreA and vreI, respectively. The process from plasmid construction to confirmation by DNA sequencing of the gene knockout was completed in one week. Furthermore, the utility of the method is highlighted in the construction of the vreA and vreI knockouts, where the start codon of vreA and the stop codon of vreI overlap. Utilizing Gibson assembly knockout mutants were constructed with single base-pair precision to generate the respective vreA and vreI knockouts while maintaining the start and stop codon of the respective genes. Overall, this method allows for the rapid construction of gene KOs in P. aeruginosa with base-pair precision.



Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.

We developed CRISPR-U™ and CRISPR-B™(based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, CRISPR-U™ and CRISPR-B™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. 


References:

Zeling Xu, Ming Li, Yanran Li, Huiluo Cao, Hua Xiang, Aixin Yan. Native CRISPR-Cas mediated in situ genome editing reveals extensive resistance synergy in the clinical multidrug resistant Pseudomonas aeruginosa. bioRxiv 496711.

Weizhong Chen, Ya Zhang, Yifei Zhang, Yishuang Pi, Tongnian Gu, Liqiang Song, Yu Wang, Quan jiangJi. iScience, Volume 6, 31 August 2018, Pages 222-231.

Huang, Weiliang, and Angela Wilks. “A rapid seamless method for gene KO in Pseudomonas aeruginosa.” BMC microbiology vol. 17,1 199. 19 Sep. 2017, doi:10.1186/s12866-017-1112-5.


Ubigene Biosciences is co-founded by biological academics and elites from China, the United States, and France. We are located in Guangzhou Science City, which serves as a global center for high technology and innovation. Ubigene Biosciences has 1000㎡ office areas and laboratories, involving genome editing, cell biology technology, and zebrafish research. We provide products and services for plasmids, viruses, cells, and zebrafish. We aim to provide customers with better gene-editing tools for cell or animal research.

We developed CRISPR-U™ and CRISPR-B™(based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, CRISPR-U™ and CRISPR-B™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. 

Genome Editing Platform
——Focusing on the Application of CRISPR-U™ and CRISPR-B™ Gene Editing Technology
1. Provides various types of gene-editing vectors for different species.
2. Provides different virus packaging services, including lentiviruses, adenoviruses and adeno-associated viruses.3. Provides high-quality services for gene knockout, point mutation and knockin cell lines. 

Cell Biology Platform
——Focusing on primary cell
1. Provides over 400 types of primary cells.
2. Provides culture strategies and related products for different cell types.3. Provides cell biology-related services such as cell isolation, extraction and validation.

Tuesday, July 7, 2020

Trichoderma Reesei using CRISPR

Lignocellulosic biomass, consisting mostly of cellulose, hemi-cellulose, and lignin, is the most abundant and renewable energy source on earth . Degradation of lignocellulosic biomass and continuation of the carbon cycle in nature is maintained mainly by microbial action, including different fungal species, such as Trichoderma, Aspergillus, and Penicillium. Microbes play important roles in nature. The biomass-degrading enzymes produced by these organisms also have applications in various fields of industry including food, fodder, paper, and textile industries. Trichoderma reesei, as one kind of fungus, is a well-known efficient producer of cellulase and hemi-cellulase, and is therefore widely employed by the enzyme industry for production of its own endogenous enzymes as well as production of heterogeneous proteins.Over the past 2 years, many studies have demonstrated that the CRISPR/Cas9 system is a powerful genome-editing method that facilitates genetic alterations in genomesin a variety of organisms. Until now, there have been no reports on the CRISPR/Cas9 system or othergenome-editing approaches in filamentous fungi, even in the model organism Neurospora crassa, despite the successful application of this technique in yeast.

Trichoderma reesei (teleomorph Hypocrea jecorina) is a mesophilic soft-rot ascomycete fungi that is widely used in industry as a source of cellulases and hemicellulases for the hydrolysis of plant cell wall polysaccharides. Lignocellulosic biomass from agricultural crop residues, grasses, wood and municipal solid waste represents an abundant renewable resource that is becoming increasingly important as a future source of biofuels. Microbes are environmentally friendly livers on the earth. Although replacement of gasoline with cellulosic ethanol may substantially reduce greenhouse gases in the atmosphere and decrease global warming, the high cost of hydrolyzing biomass polysaccharides to fermentable sugars remains a major obstacle that must be overcome before cellulosic ethanol can be effectively commercialized. As the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, much cheaper sources of these enzymes are needed. Thus, genetic engineering techniques, gene knockout protocols and DNA-mediated transformation systems have improved industrial enzyme–producing T. reesei strains.

Efficient genome editing in Trichoderma reesei using the CRISPR/Cas9 system

Researchers demonstrated the establishment of a CRISPR/Cas9 system in the filamentous fungus Trichoderma reesei by specific codon optimization and in vitro RNA transcription. It was shown that the CRISPR/Cas9 system was controllable and conditional through inducible Cas9 expression. This system generated site-specific mutations in target genes through efficient homologous recombination, even using short homology arms. This system also provided an applicable and promising approach to targeting multiple genes simultaneously. Our results illustrate that the CRISPR/Cas9 system is a powerful genome-manipulating tool for T. reesei and most likely for other filamentous fungal species, which may accelerate studies on functional genomics and strain improvement in these filamentous fungi.

Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex

Researchers tested two gene disruption methods in the fungus T. reesei using CRISPR/Cas9 in this study. The intracellularly expressed Cas9 led to unexpected off-target gene disruption in T. reesei QM9414, favoring inserting 9- or 12-bp at 70- and 100-bp downstream of the targeted ura5. An alternative method was, therefore, established by assembling Cas9 and gRNA in vitro, followed by transformation of the ribonucleoprotein complex with a plasmid containing the pyr4 marker gene into T. reesei TU-6. When the gRNA targeting cbh1 was used, eight among the twenty seven transformants were found to lose the ability to express CBH1, indicative of successful cbh1 disruption through genome editing. Large DNA fragments including the co-transformed plasmid, chromosomal genes, or a mixture of these nucleotides, were inserted in the disrupted cbh1 locus.Direct transformation of Cas9/gRNA complex into the cell is a fast means to disrupt a gene in T. reesei and may find wide applications in strain improvement and functional genomics study.


A copper-controlled RNA interference system for reversible silencing of target genes in Trichoderma reesei.

Researchers incorporated the copper-responsive tcu1 promoter into an RNAi-mediated silencing system to develop a controllable RNAi-mediated silencing system in one sort of fungus, T. reesei. As the proof-of-concept, a prototrophic pyr4 gene, highly expressed cel7a and xyr1 genes induced by Avicel and a fab1 gene, whose knockout has proved to be intractable, were successfully knocked down in the absence of copper when the respective RNAi fragment was expressed. Importantly, the phenotype of RNAi strains was shown to be reversed easily to mimic the complementation for excluding any unwanted effects resulted from the random integration of the hpRNA cassette by adding copper in the media.
we developed an RNAi-mediated silencing system driven by the P tcu1 promoter which is highly responsive to the copper ions. The developed RNAi system could readily knock down/off the target gene in the absence of copper allowing the phenotypical characterization and could mimic the complementation of the deficient strain simply by including copper in the media to exclude the unwanted effect that may result from the random integration of the hpRNA cassette. The copper-responsive RNAi-mediated silencing system is applicable on different nutritional states and represents a powerful tool for characterizing target gene functions in T. reesei.


Reference
1.Rui Liu, Ling Chen, Yanping Jiang, Gen Zou, Zhihua Zhou.A novel transcription factor specifically regulates GH11 xylanase genes in Trichoderma reesei.Biotechnology for Biofuels.2017.10:194
2.Rui Liu,Ling Chen,Yanping Jiang,Zhihua Zhou, Gen Zou,Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system.Cell Discovery.2015.1:15007
3.Zhen zhen Hao, Xiaoyun Su.Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex.BMC Biotechnology.2019.19:2
4.Diego Martinez, Randy M Berka,Bernard Henrissat Thomas,et al.Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina).Nature Biotechnology.2008.26:553-560
5.Lei Wang, Fanglin Zheng, Weixin Zhang et al.Lei Wang, Fanglin Zheng, Weixin Zhang.Biotechnology for Biofuels.2018.11:33

Multi Crispr Knockout Bacteria

  Scientists have developed a continuous  knockout cell line  technology based on the CRISPR-Cas9 system, including single and multiple (up ...