Gene-edited HuH-7 Cell Line ---- Powerful Tool for Research in Coronavirus, Drug Metabolism and Cancers

As one of the five internal organs of the human body, the liver is closely related to the body's normal metabolism, detoxification, blood coagulation, and other processes. The liver participates in the body's immunity, it is an indispensable important organ for maintaining the body's life. In the study of liver diseases, a suitable cell model is one of the important tools. However, due to the difficulties in obtaining conventional liver cells, high culture failure, and high cost of culture, the development of liver disease research has been limited. Therefore, liver cell lines with simple culture conditions and stable genetic backgrounds have become a new favorite in liver research. Huh-7 is one of the most common liver cancer lines.

 

The Huh-7 cell line was established by Nakabayshi H., and Sato, J. in 1982. It is a cancer cell line derived from a highly differentiated hepatocyte derived from the liver tumor of a 57-year-old Japanese man. Most Huh-7 cells show epithelial-like morphology and have a chromosome number between 55 and 63. Moreover, these cells are highly heterogeneous.

 

1.   Hepatitis virus research: Huh-7 is highly sensitive to the hepatitis C virus (HCV). So far, Huh-7 and its derived cell lines are the only cell lines that can effectively replicate the hepatitis C virus (HCV), so Huh-7 It is often used as a model for studying HCV. It can be used to screen drug candidates against hepatitis C virus and develop new drugs against hepatitis C virus.

 

2.  Xenograft cell model: The HuH-7 cells can be used as the CDX model of human hepatocellular carcinoma in a mouse model that enables pre-clinical tumor growth inhibition studies targeting kinase inhibitors (e.g. BZG-4000), FGFR4, anti-EGFRvIII antibodies and other novel anti-tumor growth therapeutics (e.g. sorafenib, silibinin).

 

3.  Drug research: HuH-7 cell line can be applied to study the drug efficacy and metabolism of liver cancer drugs, and to explore the molecular mechanism of drugs.

 

The combination of liver cell lines and CRISPR/Cas9 technology provides new ideas for the research of cancer, drug metabolism and coronavirus!

 

Applying CRISPR/Cas9 to knock out Huh-7's key host factors for viral replication and exploring key genes for coronavirus replication

 

CypA (cyclosporin A binding protein) is an important host factor for the replication of many RNA viruses. In addition, some studies showed that the replication of some viruses depends on CypA to varying degrees. These studies used different viruses, cell lines, and experimental designs, making it hard to compare one to another. Scientists have discovered the CypA dependence of three single-stranded sense RNA viruses that can replicate in Huh-7 cells, namely equine arteritis virus (EAV), human coronavirus (HCoV-229E) and Middle East respiratory syndrome coronavirus ( MERS-CoV). They compared the replication of these viruses, which were in the same parent Huh-7 cells or in the CypA gene knockout Huh-7 cells edited by CRISPR/Cas9 technology.

 

The sgRNAs targeting CypA, CypB, CypC, and CypD were transferred into Huh-7 cells by nuclear transfection, and positive cell pools were obtained after screening. Cyp KO Huh-7 cell pool was infected with MERS-CoV (B), HCoV-229E (C), or EAV (D), with 0.01 MOI. The plaque method was to determine virus production at 48h p.i. (B, C) or 32h p.i. (D). Among all four CypKO cell pools, the titer of MERS-CoV and HCoV-229E remained unchanged (B and C). After being infected in the Huh-7 CypA KO cell pool, the EAV virus titer decreased by 2-logs but did not vary in other knockout cell pools. The result proved that CypA did play an important role in the replication of EAV.

 

Since the Huh7 CypA-KO cell pool may have low levels of CypA expression residue, which is still sufficient to support normal levels of MERS-CoV and HCoV-229E replication. In this case, obtaining Huh7 CypA-KO monoclonals would be necessary. Different clones were chosen for target site amplification and sequencing verification, after which CypA-knockout positive clones were selected. Both wild-type Huh-7 cells and CypA-KO Huh-7 clones #1 and #2 were infected by MERS-CoV, HCoV-229E or EAV. In these two CypA-KO cell clones, the inactivation of CypA expression significantly reduced MERS-CoV replication (approximately 300%). Interestingly, in these two clones, there was no effect on HCoV-229E lacking CypA (D). However, for EAV, a ~3-log decrease in virus production (E) was observed, which showed a 10-fold stronger inhibitory effect compared to the previous Huh-7 CypA-KO cell pool.

In summary, there was no difference in the replication of MERS-CoV, HCoV-229E and EAV in the CypB-KO, CypC-KO or CypD-KO cell pools, which indicated that the replication of these viruses does not depend on CypB, CypC Or CypD in Huh-7 cells. However, similar to CypA, we cannot rule out the possibility that a very small amount of Cyp is still sufficient to support virus replication effectively. The knock-out of CypA reduced EAV production by about 3-log, while the  titers of MERS-CoV progeny decreased by about 3-fol, Also, HCoV-229E replication remained the same. This study showed that the replication of different single-stranded sense RNA viruses has significant differences in CypA dependence.

 

CRISPR/Cas9-mediated gene knockout and point mutation in Huh-7 cell model facilitate the study of the impact of different genetic differences on drug metabolism

 

In vitro studies of drug metabolism and related gene mutations often use freshly isolated or frozen human/animal liver cells; however, primary liver cells are probably not the best choice because they require liver collection and are expensive. Additionally, primary liver cells are not immortalized , which leads to large differences between different kinds of the cells. In most cases, the cell lines are identical in genome. Therefore, researchers developed a CRISPR/Cas9 modified human hepatocyte cell line so as to continuously study the effects of genetic variation on drug metabolism. To study the effects of CYP3A5 mutations on the metabolism of two enzyme substrates, the sedative or anesthetic midazolam (MDZ) and the immunosuppressant tacrolimus (Tac).

About 50% of oral drugs are metabolized by CYP3A4 and CYP3A5, which are the most abundant in the liver have highly variable expression. The loss of function of CYP3A5*3 (rs776746) allele is very common among Caucasians. Tac has a comparatively lower metabolic rate than those with CYP3A5*1 genotype. However, the CYP3A5*1 allele is abundant in African Americans who have rapid metabolism of MDZ, Tac, and other drugs. Therefore, CYP3A5 genotype is vital to determine the appropriate dose of drugs.

To present, no commercial liver cell line has been diploid on chromosome 7 and can express CYP3A5*1. Huh-7 cell line can convert substrate MDZ to its metabolite hydroxylated 1-OH-MDZ and 4-OH-MDZ through CYP3A4 activity, though they are not effective in the metabolism of MDZ because of their identity as homozygote to the CYP3A5*3 allele. Therefore, it is necessary to develop a liver cell line that can simulate the rapid metabolic process of drugs that are related to the CYP3A5*1 genotype.

The gRNA, Cas9, and ssODN were co-transformed into Huh-7 cells by nuclear transfection method, followed by monoclonal selection. Different clones were selected for target site amplification and sequencing verification, while positive clones with gene knockout were selected. By knocking out or point-mutating the splice junction of the Exon-3B of CYP3A5*3, three CYP3A5*1 cell lines were obtained.

Compared with WT  Huh-7, CYP3A5*1/*3sd (heterozygous KO), CYP3A5*1/*1dd (homozygous KO) or CYP3A5*1/*3pm (point mutation) express CYP3A5*1 mRNA, Both CYP3A5 mRNA and protein expression increased. Therefore, through the CRISPR/Cas9 technology, the *3 genotype was successfully transformed into the *1 genotype, thereby activating the expression of CYP3A5 in the Huh-7 cell line. This cell model can accelerate preclinical drug development, save time and money, and more accurately predict drug metabolism, pharmacokinetics, toxicity, and efficacy in different populations or genotypes.

 

Using CRISPR/Cas9 to knockin GFP to the Nanog in Huh-7 cells, revealing the reasons for gender differences in the incidence of hepatocellular carcinoma

 

Hepatocellular carcinoma (HCC) is a common malignant tumor, and its morbidity has obvious gender differences ---- the inclination to men. Studies have shown that the androgen/androgen receptor signal axis is related to the incidence of various hormone-related tumors such as prostate cancer and cervical cancer. As an important place for androgen metabolism, the liver has a high level of androgen in its microenvironment. Hepatocellular carcinoma seems to have a close relationship with the androgen/androgen receptor signal axis.

Cancer Stem Cells (CSCs) are closely related to the occurrence and metastasis of tumors. Their ability to self-renewal and unlimited proliferation is a key factor in cancer development. Studies have revealed that the pluripotency factor Nanog is involved in maintaining the dryness of CSCs. However, it is unclear whether the androgen/androgen receptor signal axis affects the dry maintenance of HCC cells through Nanog-related pathways.

The researchers found that the expression of the androgen receptor is very high in liver cancer tissues and is related to Nanog. Subsequently, the endogenous Nanog of huh7 cells was labeled with GFP by CRISPR/Cas9, which confirmed the co-localization of the androgen receptor and Nanog in HCC cells. The gRNA, Cas9, and Donor were co-transformed into huh-7 cells by nuclear transfection, followed by drug screening and monoclonal cultures selection. Different clones were chosen for target site amplification and sequencing, after which positive clones with homozygous knock-in were selected.

Through subsequent in vitro experiments, the researchers proved that the signal axis can promote the stemness of HCC cells, which would be achieved in a Nanog-dependent manner. By activating the transcription, this effect can be blocked by androgens Or AR degradation enhancer.

 

These studies show that the androgen/androgen receptor signaling axis provides evidence for the inhibition of this axis in HCC therapy by affecting the stemness of tumor cells, which also offers a possible way for the suppression of axons in the treatment of liver cancer.

 

CRISPR-U™ efficiently modify genes in liver cell lines

 

CRISPR-U ™ is an exclusive technology independently developed by Ubigene Bioscience for gene editing cell lines. By optimizing gene editing vectors and processes, the efficiency in gene-cutting and recombination of CRISPR-U ™ is 10 times higher than the conventional CRISPR/Cas9 technology. We are capable of customizing genetically-modified (KO, KI and point mutation) liver cell lines as you desire and satisfying various needs in gene editing.

 

References：

1.       de Wilde A H, Zevenhoven-Dobbe J C, Beugeling C, et al. Coronaviruses and arteriviruses display striking differences in their cyclophilin A-dependence during replication in cell culture[J]. Virology, 2018, 517: 148-156.

2.       Dorr C R, Remmel R P, Muthusamy A, et al. CRISPR/Cas9 genetic modification of CYP3A5* 3 in HuH-7 human hepatocyte cell line leads to cell lines with increased midazolam and tacrolimus metabolism[J]. Drug Metabolism and Disposition, 2017, 45(8): 957-965.

3.       Jiang L, Shan J, Shen J, et al. Androgen/androgen receptor axis maintains and promotes cancer cell stemness through direct activation of Nanog transcription in hepatocellular carcinoma[J]. Oncotarget, 2016, 7(24): 36814.

CRISPR Editing Aspergillus fumigatu with High Efficiency and Target Rate

Introduction:

Aspergillus fumigatus is a species of fungus in the genus Aspergillus and is one of the most common Aspergillus species to cause disease in individuals with immunodeficiency. It can be found throughout the environment, including in soil, plant matter, and household dust. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes. Thus, A. fumigatus was considered for years to be a weak pathogen. Nowadays, Aspergillus fumigatus becomes more and more popular in research because this specie plays an important role in the environment and human life. Also, CRISPR/Cas9 is a novel genome-editing system that has been successfully established in Aspergillus fumigatus. Scientists have generated a CRISPR system for Aspergillus fumigatus to study its function by gene knockout, gene knockin, point mutation, etc.

Aspergillus fumigatus is one of the most common causes of aspergillosis. The first one is allergic bronchopulmonary aspergillosis, it is an allergic reaction to the Aspergillus spores. This reaction can lead to damage in your airways and lungs. It is often found in people that have conditions such as asthma and cystic fibrosis. The second one is Chronic pulmonary aspergillosis, which can occur in people with chronic lung conditions that cause air spaces called cavities to form in the lung. The third one is invasive aspergillosis, it is the most severe form of aspergillosis and can be fatal if not treated. It occurs when an aspergillosis infection begins in the lungs and spreads to other parts of the human body, such as skin, brain, or kidney. Invasive aspergillosis occurs only in people who have a severely weakened immune system.

Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

To test the CRISPR/Cas9 system’s feasibility for targeted gene disruption in A. fumigatus. As a proof of principle, researchers first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. Researchers further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen.

 

Using CRISPR to Gene Manipulation in Aspergillus fumigatus

The current state of the technology relies heavily on DNA-based expression cassettes for delivering Cas9 and the guide RNA (gRNA) to the cell. Therefore, the power of technology is limited to strains that are engineered to express Cas9 and gRNA. To overcome such limitations, researchers developed a simple and universal CRISPR-Cas9 system for gene deletion that works across different genetic backgrounds of A. fumigatus. The system employs in vitro assembly of dual Cas9 ribonucleoproteins (RNPs) for targeted gene deletion. Additionally, the CRISPR-Cas9 system utilizes 35 to 50 bp of flanking regions for mediating homologous recombination at Cas9 double-strand breaks (DSBs). Similar deletion efficiencies were obtained in the clinical isolate DI15-102. The data shows that in vitro-assembled Cas9 RNPs coupled with microhomology repair templates are an efficient and universal system for gene manipulation in A. fumigatus. In this study, tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. The simple and universal CRISPR-Cas9 system for gene manipulation in this study generates efficient gene targeting across different genetic backgrounds of A. fumigatus.

 

Ubigene developed CRISPR-B™  which optimizes the microbial gene-editing vectors and processes. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Easily achieve microbial gene knockout (KO), point mutation (PM), and knockin (KI).

 

References:

Latgé JP. Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev. 1999;12(2):310-350

Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

Kevin K. Fuller, Shan Chen, Jennifer J. Loros, Jay C. Dunlap

Eukaryotic Cell Oct 2015, 14 (11) 1073-1080

A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates

Qusai Al Abdallah, Wenbo Ge, Jarrod R. Fortwendel

mSphere Nov 2017, 2 (6) e00446-17; DOI: 10.1128/mSphere.00446-17

CRISPR Editing Fusarium with High Efficiency and Target Rate

Fusarium is a large cosmopolitan genus of imperfect fungi and is of interest primarily because numerous species are important plant pathogens (Nelson et al. 1981), produce a wide range of secondary metabolites, and/or cause opportunistic infections in humans. Although Fusarium research over the past 100 years has advanced our understanding of this important group of fungi, many aspects of its biology still need to be addressed. That’s why researchers are interested in conducting gene-editing in Fusarium including knockout, knock-in and point mutation, etc.

 

Most Fusarium species are soil fungi and have a worldwide distribution. Some are plant pathogens, causing root and stem rot, vascular wilt, or fruit rot. Several species have emerged as important opportunistic pathogens in humans causing hyalohyphomycosis (especially in burn victims and bone marrow transplant patients), mycotic keratitis, and onychomycosis (Guarro 2013). Other species cause storage rot and are important mycotoxin producers. Gene knockout in Fusarium services might be different from those in cell lines. Gene-editing such as CRISPR in Fusarium will be more complicated but will also bring many benefits to human life.

 

CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum

Fusarium oxysporum is an economically important pathogen that widely exists in the environment and is capable of causing serious problems in crop production and animal/human health. One important step for the characterization of a fungal protein with an unknown function is to determine its subcellular localization within the cell. To facilitate the study of important functional regulators or key virulence factors, a CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies was developed, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). The results indicate that this EGT system is efficient and can be another molecular tool to resolve the function(s) of proteins and infection strategies of F. oxysporum.

 

Generation of Fusarium graminearum Knockout Mutants by the Split-marker Recombination Approach

Fusarium graminearum is a destructive phytopathogen and shows an impressive metabolic diversity. Gene deletion is an important and useful approach for gene function study. Here a protocol for generating gene deletion mutant by applying a “split-marker” deletion strategy (Catlett et al., 2003) with PEG-mediated protoplast transformation (Yuan et al., 2008; Martín, 2015) was present. For generating single-gene deletion mutants, a variety of genes conferring resistance to antibiotics are available, e.g., genes resistant to Hygromycin B, Geneticin/G418, Bialaphos/Phosphothricin, Nourseothricin, Blasticidin, and Phleomycin. Among these, the gene resistant to Hygromycin B is the most widely used. In this protocol, HPH worked as a resistant gene. Other markers can also be chosen according to the experiment as well (Turgeon et al., 2010).

 

Biochemical Characterization and Knockout Mutant In Fusarium graminearum

F. graminearum and other species can produce auxin, and auxin levels are increased in Fusarium infected plants (Kidd et al., 2011; Wang et al., 2018). Since it had been reported that ethylene insensitivity in transgenic wheat increased Fusarium resistance and reduced the content of the mycotoxin deoxynivalenol (DON) in infected wheat, researchers generated single and double knockout mutants of both genes in the F. graminearum strain PH-1.

Six PCR confirmed double-knockout strains were chosen for the virulence test with 10 replicates per strain. The progress of infection was observed over 16 days followed by the analysis of DON and D3G after harvesting. No statistically significant effect of the gene disruptions on the fungal spread or mycotoxin content was detected, indicating that the ability of the fungus to manipulate the production of the gaseous plant hormones ethylene and H2S is dispensable for full virulence.

 

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. Easily achieve microbial gene knockout (KO), point mutation (PM), and knockin (KI).

 

References:

CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum. Qiang Wang, Jeffrey J. Coleman. Fungal Genetics and Biology, Volume 126, May 2019, Pages 17-24.

Wang, W., and Tang, W. (2018). Generation of Fusarium graminearum Knockout Mutants by the Split-marker Recombination Approach. Bio-101: e2976.

Svoboda T, Parich A, Güldener U, Schöfbeck D, Twaruschek K, Václavíková M, Hellinger R, Wiesenberger G, Schuhmacher R and Adam G (2019) Biochemical Characterization of the Fusarium graminearum Candidate ACC-Deaminases and Virulence Testing of Knockout Mutant Strains. Front. Plant Sci. 10:1072. doi: 10.3389/fpls.2019.01072

 

Applying CRISPR in Yeasts | Gene-knockout, knock-in, etc.

Introduction:

 

Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. The yeast species Saccharomyces cerevisiae converts carbohydrates to carbon dioxide and alcohols in a process known as fermentation. The products of this reaction have been used in baking and the production of alcoholic beverages for thousands of years. S. cerevisiae is also an important model organism in modern cell biology research, and is one of the most thoroughly studied eukaryotic microorganisms. Researchers have cultured it in order to understand the biology of the eukaryotic cell and ultimately human biology in great detail. Other species of yeasts, such as Candida albicans, are opportunistic pathogens and can cause infections in humans. Yeasts have recently been used to generate electricity in microbial fuel cells and to produce ethanol for the biofuel industry.

 

The useful physiological properties of yeast have led to their use in the field of biotechnology. Fermentation of sugars by yeast is the oldest and largest application of this technology. Many types of yeasts are used for making many foods: baker's yeast in bread production, brewer's yeast in beer fermentation, and yeast in wine fermentation and for xylitol production. So-called red rice yeast is actually a mold, Monascus purpureus. Yeasts include some of the most widely used model organisms for genetics and cell biology.

The methods based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system have quickly gained popularity for genome editing and transcriptional regulation in many organisms, including yeast.

 

Red/ET recombination system is a classical method of microbial gene editing, which can achieve the knockin, knockout, point mutation and other modifications of the target gene. This technology has been widely used in genetic modification of genomic DNA, such as bacterial artificial chromosome(BAC), Escherichia coli chromosome. However, the efficiency of this system still needs to be improved. How to improve the efficiency of gene recombination and editing has always been a hotspot of microbial gene editing. Therefore, CRISPR/Cas9 technology is adopted to improve the efficiency of microbial gene editing. The price for gene editing yeast services will be similar to those in cell lines.

 

CRISPR/Cas9 is an acquired immune system in bacteria and archaea and can be used to fight against invading viruses and exogenous DNA. In recent years, the CRISPR/Cas9 gene-editing technology has been widely used because it is simple and efficient. It has been the most advanced method for gene editing. 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.

 

Genome-editing in Yeast using precise targeting CRISPR

 

When Cas9 protein and gRNA are expressed in yeast cells, Cas9 introduces DSBs that must be repaired by the cells via non-homologous end joining (NHEJ) or homologous recombination (HR). By supplying a DNA repair template for use in HR, various DNA modifications can be obtained. In the case of efficient cutting, the generated DSBs serve as a negative selection. Thus, there is no need for using a selective marker as in non-CRISPR genome editing methods. Relatively precise and flexible targeting and elimination of the need for positive selection are the two key advantages of the CRISPR/Cas9 technology for yeast genome engineering.

 

The rrk1 CRISPR-Cas9 method enables rapid and efficient genome manipulation and unlocks the CRISPR toolset for use in fission yeast

Application of the CRISPR-Cas9 genome editing system in the model organism  has been hampered by the lack of constructs to express RNA of arbitrary sequence. Therefore, researchers present expression constructs that use the promoter/leader RNA of K RNA (rrk1) and a ribozyme to produce the targeting guide RNA. Together with constitutive expression of Cas9, this system achieves selection-free specific mutagenesis with efficiencies approaching 100%. The fission yeast Schizosaccharomyces pombe has proven to be a useful model organism because of its higher degree of similarity to genomes of higher eukaryotes than the classic yeast model S. cerevisiae. S. pombe remains less well studied than S. cerevisiae, and lags behind in availability of molecular tools. In particular, lack of a portable RNA Pol III promoter to express sgRNA has prevented the implementation of the CRISPR-Cas9 system. A CRISPR-Cas9 system that enables specific, precise and efficient genome editing in S. pombe is developed. Its flexibility enables the use in fission yeast of the full range of CRISPR-derived tools for genome manipulation.

 

Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in  yeast

The recombination machinery in P. pastoris is less effective in contrast to Saccharomyces cerevisiae, where efficient homologous recombination naturally facilitates genetic modifications. The lack of simple and efficient methods for gene disruption and specifically integrating cassettes has remained a bottleneck for strain engineering in P. pastoris. Therefore tools and methods for targeted genome modifications are of great interest. Researchers developed a CRISPR/Cas9 system that enables specific and precise genome engineering in P. pastoris as a potent alternative to the currently applied genome engineering strategies.

The establishment of CRISPR/Cas9 technologies for P. pastoris and demonstrate targeting efficiencies approaching 100%. However there appeared to be a narrow window of optimal conditions required for efficient CRISPR/Cas9 function for this host. Scientists systematically tested combinations of various codon optimized DNA sequences of CAS9, different gRNA sequences, RNA Polymerase III and RNA Polymerase II promoters in combination with ribozymes for the expression of the gRNAs and RNA Polymerase II promoters for the expression of CAS9. Only 6 out of 95 constructs were functional for efficient genome editing.

This optimized CRISPR/Cas9 system for gene disruption studies, was used to introduce multiplexed gene deletions and to test the targeted integration of homologous DNA cassettes. This system allows rapid, marker-less genome engineering in P. pastoris enabling unprecedented strain and metabolic engineering 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. Easily achieve microbial gene knockout (KO), point mutation (PM) and knockin (KI).

 

References:

Vratislav Stovicek, Carina Holkenbrink, Irina Borodina, CRISPR/Cas system for yeast genome engineering: advances and applications, FEMS Yeast Research, Volume 17, Issue 5, August 2017, fox030, https://doi.org/10.1093/femsyr/fox030

Jacobs, J., Ciccaglione, K., Tournier, V. et al. Implementation of the CRISPR-Cas9 system in fission yeast. Nat Commun 5, 5344 (2014). https://doi.org/10.1038/ncomms6344

Astrid Weninger, Anna-Maria Hatzl, Christian Schmid, Thomas Vogl, Anton Glieder,

Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris, Journal of Biotechnology, Volume 235, 2016, Pages 139-149, ISSN 0168-1656.

 

Gene-editing Aspergillus Niger using CRISPR | High-efficiency & low off-target rate

Introduction:

Aspergillus niger is a fungus and one of the most common species of the genus Aspergillus. It causes a disease called "black mold" on certain fruits and vegetables such as grapes, apricots, onions, and peanuts, and is a common contaminant of food. It is ubiquitous in soil and is commonly reported from indoor environments, where its black colonies can be confused with those of Stachybotrys (species of which have also been called "black mold").

Aspergillus niger causes sooty mold of onions and ornamental plants. The infection of onion seedlings by A. niger can become systemic, manifesting only when conditions are conducive. A. niger causes a common postharvest disease of onions, in which the black conidia can be observed between the scales of the bulb. The fungus also causes disease in peanuts and grapes.

Aspergillus niger is less likely to cause human disease than some other Aspergillus species. In extremely rare instances, humans may become ill, but this is due to serious lung disease, aspergillosis, that can occur. Aspergillosis is, in particular, frequent among horticultural workers who inhale peat dust, which can be rich in Aspergillus spores. The fungus has also been found in the mummies of ancient Egyptian tombs and can be inhaled when they are disturbed. A. niger is one of the most common causes of otomycosis (fungal ear infections), which can cause pain, temporary hearing loss, and, only in severe cases, damage to the ear canal and tympanic membrane.

Some strains of A. niger have been reported to produce potent mycotoxins called ochratoxins;  other sources disagree, claiming this report is based upon a misidentification of the fungal species. Recent evidence suggests some true A. niger strains do produce ochratoxin A. It also produces the isoflavone orobol.

The filamentous fungus Aspergillus niger is an important cell factory used in the industry for the production of enzymes and organic acids. Owing to its genetic tractability, A. niger is widely used for research in fungal physiology, biochemistry, and biotechnology. The availability of the genome sequence of this organism has facilitated numerous studies in gene function, gene regulation, primary and secondary metabolism. The utility of A. niger as an industrial cell factory and as a model organism for research can be further improved by the development of a high-efficiency genome-editing system. Aspergillus niger is an important industrial producer of enzymes due to its high capacity for producing exocellular secretory proteins. The CRISPR/Cas9 system has been developed as a genetic manipulation tool in A. niger services. However, the prices for gene knockout aspergillus niger are similar to those for cell lines.

 

Engineering Aspergillus niger for galactaric acid production

The use of CRISPR/Cas9 mediated gene deletion technology in A. niger in a metabolic engineering application. A transcriptomics approach was used to identify genes involved in galactaric acid catabolism. Several genes were deleted using CRISPR/Cas9 together with in vitro synthesized sgRNA. As a result, galactaric acid catabolism was disrupted. An engineered A. niger strain combining the disrupted galactaric and D-galacturonic acid catabolism with an expression of a heterologous uronate dehydrogenase produced galactaric acid from D-galacturonic acid. The resulting strain was also converting pectin-rich biomass to galactaric acid in a consolidated bioprocess. As a result, a strain for the efficient production of galactaric acid from D-galacturonic acid was generated.

 

Engineering gRNA promoter using CRISPR/Cas9 in Aspergillus niger

In eukaryotes, search and optimization of a suitable promoter for guide RNA expression is a significant technical challenge. Researchers used the industrially important fungus, Aspergillus niger, to demonstrate that the 5S rRNA gene, which is both highly conserved and efficiently expressed in eukaryotes, can be used as a guide RNA promoter. The gene-editing system was established with 100% rates of precision gene modifications among dozens of transformants using short (40-bp) homologous donor DNA. This system was also applicable for the generation of designer chromosomes, as evidenced by the deletion of a 48 kb gene cluster required for the biosynthesis of the mycotoxin fumonisin B1. Moreover, this system also facilitated the simultaneous mutagenesis of multiple genes in A. niger. We anticipate that the use of the 5S rRNA gene as a guide RNA promoter can broadly be applied for engineering highly efficient eukaryotic CRISPR/Cas9 toolkits. Additionally, the system reported here will enable the development of designer chromosomes in the model and industrially important fungi.

 

Performing CRISPR mutagenesis in Aspergillus using extra-chromosomal plasmid

With the ability of nucleases to make DNA double-strand break (DSB) in the host organism, nuclease-based gene targeting methods have been successfully used for gene disruption, knock-in mutation as well as improving heterologous protein production by integrating foreign genes to defined genomic loci. An efficient promoter that can facilitate gRNA transcription in vivo is a bottleneck of the adoption of the CRISPR/Cas9 system in Aspergilli. The method of delivering guide RNA (gRNA) remains a bottleneck in performing CRISPR mutagenesis in Aspergillus species. Researchers reported a gRNA transcription driven by endogenous tRNA promoters which include a tRNA gene plus 100 base pairs of upstream sequence. Co-transformation of a cas9-expressing plasmid with a linear DNA coding for gRNA demonstrated that 36 of the 37 tRNA promoters tested were able to generate the intended mutation in A. niger. When gRNA and cas9 were expressed in a single extra-chromosomal plasmid, the efficiency of gene mutation was as high as 97%. The results demonstrate that tRNA promoter-mediated gRNA expressions are reliable and efficient in genome editing in A. niger.

 

Improving targeting efficiency in Aspergillus niger based on in vitro assembled ribonucleoproteins

In this study, a CRISPR/Cas9 facilitated transformation and genome editing method based on in vitro assembled ribonucleoprotein complexes was developed for the filamentous fungus Aspergillus niger. The method was downscaled to be compatible with 96-well microtiter plates. The optimized method resulted in 100% targeting efficiency for a single genomic target. After the optimization, the method was demonstrated to be suitable for multiplexed genome editing with two or three genomic targets in a metabolic engineering application. As a result, an A. niger price strain with improved capacity to produce galactarate, a potential chemical building block, was generated.

The developed microtiter plate compatible CRISPR/Cas9 method provides a basis for high-throughput genome editing workflows in A. niger and other related species. Besides, it improves the cost-effectiveness of CRISPR/Cas9 genome editing methods in fungi based on in vitro assembled ribonucleoproteins. The demonstrated metabolic engineering example with multiplexed genome editing highlights the applicability of the method.

 

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. Easily achieve microbial gene knockout (KO), point mutation (PM) and knockin (KI).

 

References:

1.       Samson RA, Houbraken J, Summerbell RC, Flannigan B, Miller JD (2001). "Common and important species of fungi and actinomycetes in indoor environments". Microorganisms in Home and Indoor Work Environments. CRC. pp. 287–292. ISBN 978-0415268004.

2.       Kuivanen, J., Wang, Y.J. & Richard, P. Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9. Microb Cell Fact 15, 210 (2016).

3.       Xiaomei Zheng, Ping Zheng, Kun Zhang, Timothy C. Cairns, Vera Meyer, Jibin Sun, and Yanhe Ma. 5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger. ACS Synth. Biol. 2019, 8, 7, 1568–1574.

4.       Song L, Ouedraogo JP, Kolbusz M, Nguyen TTM, Tsang A. Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger. PLoS One. 2018;13(8):e0202868. Published 2018 Aug 24.

5.       Kuivanen, J., Korja, V., Holmström, S. et al. Development of microtiter plate scale CRISPR/Cas9 transformation method for Aspergillus niger based on in vitro assembled ribonucleoprotein complexes. Fungal Biol Biotechnol 6, 3 (2019).

 

ACE2 knockout cell line | high-efficiency and low off-target rate

Angiotensin-converting enzyme (ACE) is a kind of metalloproteinase encoding 805 amino acids, located in X chromosome (Xp22.2). ACE is a type I transmembrane glycoprotein with a single extracellular catalytic domain that plays an important regulatory role in the renin-angiotensin system (RAS). ACE2, a homolog of ACE, can split Ang II into Ang (1-7) polypeptide, which has anti-inflammatory functions: protecting cardiomyocytes, relaxing blood vessels, anti-proliferation, and can enhance the activity of bradykinin (an inflammatory mediator).

Angiotensin-converting enzyme 2 (ACE2) is a type 1 integral membrane glycoprotein that is expressed and active in most tissues. Also, it shares some homology with angiotensin-converting enzyme (ACE) but is not inhibited by ACE inhibitors.

 

ACE2 expression restrict virus replication in human cells

It was isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells that efficiently binds the S1 domain of the SARS-CoV S protein. It has been reported that ACE2 is the main host cell receptor of 2019-nCoV and plays a crucial role in the entry of virus into the cell to cause the final infection. It was recently shown that murine ACE2 does not allow for efficient SARS-CoV replication, raising the possibility of alternate receptor use in non-primate cells.

Cell lines were infected with the pseudotyped retrovirus expressing ACE2. At 24 to 48 h after lipofection or 72 h after retrovirus infection, cells were infected with SARS-CoV. ACE2 expression from the pseudotyped retrovirus resulted in SARS-CoV replication in all cell lines examined, including those still refractory following plasmid ACE2 expression. Based on these results, the in vitro host range of SARS-CoV is primarily determined by the presence of its receptor, ACE2.

The 293T cells were unable to support virus replication after plasmid ACE2 expression despite high levels of ACE2 expression. However, ACE2 expression from the pseudotyped retrovirus resulted in efficient virus replication in 293T cells. Similar observations were made in A549 and AK-D cells. Therefore, restriction of SARS-CoV replication in these cells was recently shown to be overcome by expression of human ACE2.

 

The importance of ACE2 in maintaining the balance of the RAS system

The main role of ACE2 is the degradation of Ang II resulting in the formation of angiotensin 1–7 (Ang 1–7) which opposes the actions of Ang II. Increased Ang II levels are thought to upregulate ACE2 activity, and in ACE2 deficient mice Ang II levels are approximately double that of wild-type mice, whilst Ang 1–7 levels are almost undetectable. Thus, ACE2 plays a crucial role in the RAS because it opposes the actions of Ang II. Consequently, it has a beneficial role in many diseases such as hypertension, diabetes, and cardiovascular disease where its expression is decreased.  Current therapeutic strategies for ACE2 involve augmenting its expression using ACE2 adenoviruses, recombinant ACE2 or compounds in these diseases thereby affording some organ protection.

 

ACE2 helps research in Kidney Disease

Kidney injury is largely mediated by Ang II. Some studies have shown that ACE2 gene knockout can lead to an increase in blood pressure, glomerular damage, and renal fibrosis in diabetic mice. Exogenous human recombinant ACE2 (hACE2) can slow down the progress of diabetic nephropathy (DKD) by reducing albumin excretion. ACE2 was highly expressed in kidney, mainly in brush border cells of proximal renal tubules, endothelial cells, smooth muscle cells of renal vessels, and podocytes. It has been reported that in damaged renal tubules, the increase of Ang II may be a possible mediator for further renal damage in human renal diseases. Therefore, the imbalance between ACE and ACE2 in the kidney followed by high level of Ang II may lead to renal damage. Hypertensive nephropathy is a common complication of hypertension, with its main mechanisms being inflammation associated with Ang II, oxidative stress, and renal fibrosis. It is worth noting that in April 2019, the school of medicine of Jilin University found that Ginsenoside Rg3 can alleviate the Ang II-mediated renal injury in rats and mice by upregulating ACE2 in renal tissue. In addition to anti-tumor activity, Rg3 can also protect the cardiovascular system through various mechanisms, including anti-inflammatory, anti-oxidative stress, and anti-fibrosis.

 

ACE2 deletion resulted in modest elevations in systolic blood pressure levels

ACE2 is widely expressed in cardiomyocytes, cardiac fibroblasts, and coronary artery endothelial cells. ACE2 is an important regulatory protein in RAS, the system which regulates the balance of body fluid and blood pressure and maintains the tension of blood vessels. The overactivation (increase of vasoconstriction) or depletion (decrease of vasodilation) of RAS will lead to vascular dysfunction, which is the main cause of atherosclerosis and cardiovascular disease (CVD). 

The renin-angiotensin system (RAS) has been implicated in atherosclerotic lesions and progression to chronic kidney diseases. We examined regulatory roles of angiotensin-converting enzyme 2 (ACE2) in the apolipoprotein E (ApoE) knockout (KO) kidneys. Downregulation of ACE2 and nephrin levels was observed in ApoEKO kidneys. Genetic ACE2 deletion resulted in modest elevations in systolic blood pressure levels and Ang II type 1 receptor expression and reduced nephrin expression in kidneys of the ApoE/ACE2 DKO mice with a decrease in renal Ang-(1-7) levels. 

 

ACE2 knockout mouse

The first description of an ACE2 knockout mouse line by Crackower and associates suggested that ACE2 plays an essential role in regulating normal cardiac function in vivo. In this mouse line, a null mutation was generated by replacing portions of exons 7–9 of the Ace2 gene with a neomycin cassette in the antisense orientation; exon 9 encodes the zinc‐binding (HEMGH) motif of the enzyme (Donoghue et al. 2000; Tipnis et al. 2000). The dominant phenotype of these ACE2‐deficient mice was a marked defect in cardiac contractility. This decrease in left ventricular systolic function, documented by echocardiography in anaesthetized mice, was more severe in older, male ACE2‐deficient mice and was accompanied by reduced blood pressures. In 6‐month‐old male mice, left ventricular fractional shortening was reduced by as much as 40%. Additionally, cardiac structure was abnormal, with wall thinning and enlarged cardiac chambers. However, there was no indication of cardiac hypertrophy or fibrosis in the ACE2‐deficient mice, and overall heart weights were very similar between ACE2 knockout and wild‐type animals.

 

References:

1.      Mossel EC, Huang C, Narayanan K, Makino S, Tesh RB, Peters CJ. Exogenous ACE2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication. J Virol. 2005;79(6):3846‐3850.

2.      Li, W., Moore, M., Vasilieva, N. et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450–454 (2003).

3.      Xu, H., Zhong, L., Deng, J. et al. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int J Oral Sci 12, 8 (2020).

4.      Tikellis C, Thomas MC. Angiotensin-Converting Enzyme 2 (ACE2) Is a Key Modulator of the Renin Angiotensin System in Health and Disease. Int J Pept. 2012;2012:256294.

5.      Angiotensin‐converting enzyme 2 gene targeting studies in mice: mixed messages. Susan B. Gurley  Thomas M. Coffman. Experimental PhysiologyVolume 93, Issue 5.

CRISPR-U™ Knockout stable Cell line | High efficiency

CRISPR-U™ (based on CRISPR/Cas9 technology), developed by Ubigene, is more efficient than general CRISPR/Cas9 in double-strand breaking, and CRISPR-U™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. With CRISPR-U, Ubigene has successfully edit genes on more than 100 cell lines.

CRISPR/Cas9 recognizes the target sequence with gRNA, and guide Cas9 endonuclease to cut the upstream of PAM, resulting in the double-strand break (DSB) of the target site DNA. To repair the DSB, the cell uses its own DNA repair mechanism to add or delete or replace pieces of DNA sequences via Homology Directed Repair (HDR) or Non-Homologous End Joining (NHEJ).

CRISPR-U™ Point mutation Cell line | High efficiency

CRISPR/Cas9 and ssODN used to repair the point mutation in A79V-hiPSC. A) The genomic sequence surrounding the mutation site: mutated nucleotide (T, red); sgRNA recognition site containing 20 bp (yellow); CRISPR cutting site between the 17th and 18th bp (bold); forward and reverse primers (pink). B) ssODN with 120 bp, 60 bp upstream and 60 bp downstream the mutation site containing the WT nucleotide (C, green).

Virus Packaging - AAV for KO, KI and over-expression in vitro and in vivo

Adeno-associated virus (AAV) is a kind of Parvovirus, which genome is single-stranded DNA. It can infect both divisive and non-divisive cells. Adenoviruses or herpes viruses are usually needed to assist AVV to replicate in vivo. Recombinant AAV (rAAV) is a viral vector that combines the AAV2 genome with capsid protein genomes of different serotypes. CDS or RNAi interference sequences of the target genes can be inserted into the rAAV plasmid. AAV is mainly used in vivo injection because it has low immunogenicity and long-term expression.

Ubigene's AAV will be purified by ultracentrifugation, and its genome copy will be titrated by qPCR. The titer of AAV ranged from 10^12 to 10^13 v. g. / ml.

Virus Packaging - Adenovirus for KO, KI and over-expression in vitro and in vivo

Adenovirus is a linear and double-stranded DNA virus that without a surface envelope. It can infect both divisive and non-divisive cells. Recombinant adenovirus can enter cells through receptor-mediated endocytosis, while its genome is not integrated with the host cell genome. Adenovirus is widely used in the researches of the cardiovascular, liver, muscle, lung, cancer, and other fields. It has the characteristics of wide host range, strong immunogenicity, large capacity, no integration with the genome, transient expression, and so on.

To ensure the safety, human adenovirus type 5 (Ad5), which is a replication-incompetent (E1 and E3 genes deleted) adenovirus, is used. E1 is essential for the replication of adenovirus. The absence of E1 prevents its self-replication. Its replication depends on packaging cells such as 293A cells, which provide trans-complementary replication for adenovirus. The protein expressed by the E3 gene can resist the host's antiviral defense system, and the removal of the E3 region can reduce the host's immune response.

The adenovirus particles provided by Ubigene would be centrifuged and filtered by gradient ultracentrifugation, and the titer would be determined by chitin immunoassay. The titer of adenovirus is in the range of 10 ^ 10 ~ 10 ^ 12pfu / ml.

Virus Packaging - Lentivirus for KO, KI and over-expression in vitro and in vivo

Ubigene provides lentivirus, adenovirus, and adeno-associated virus (AAV) with different sizes and grades of purification. Our products can easily achieve knockout (KO), overexpression and knockdown in vitro and in vivo.