Cloning Services - Point mutation Plasmid | Precise and High efficiency

CRISPR/Cas9 system is a defense mechanism used by bacteria to resist the invasion of viruses and foreign plasmids. At present, the type II CRISPR/Cas9 system is the most developed and widely used system. It 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 sequence via Homology Directed Repair (HDR) or Non-Homologous End Joining (NHEJ). 

Cloning Services - Knockin Plasmid | Precise and High efficiency

CRISPR/Cas9 system is a defense mechanism used by bacteria to resist the invasion of viruses and foreign plasmids. At present, the type II CRISPR/Cas9 system is the most developed and widely used system. It 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 a DNA sequence via Homology Directed Repair (HDR) or Non-Homologous End Joining (NHEJ).

Guide RNA and Cas9 complex cause a double-strand break (DSB) on the target site of DNA. The donor vector carrying knockin sequence is the template for homologous recombination repair (HDR), and it recombines to the target site.

Cloning Services - Knockdown Plasmid | Precise and High efficiency

RNA interference (RNAi) is induced by a highly conserved, double-stranded RNA (dsRNA), which would cause specific degradation of homologous mRNA. Specific genes can be silenced, resulting in the loss of function or suppression of gene expression.

Note: Ubigene provides customized plasmid solutions, including different types of backbone, expression system, shRNA design, and Pol II promoters (Mir30-shRNA), etc.

Cloning Services - Overexpression Plasmid | Precise and High efficiency

The target coding sequence (CDS) was cloned into a plasmid, and promoter on plasmid regulates the expression of the target gene. In addition, the reporter gene and selection marker can be used for screening. Ubigene developed a series of YOE plasmids, including lentivirus plasmids, AAV plasmids, and non-viral plasmids, which can be widely used in vitro or in vivo.

Note：Ubigene provides customized plasmid solutions. In addition to the selection of the above-mentioned plasmid backbone, customers can also choose different promoters: constitutive/specific/inducible promoter, the selection of connection mode between multiple target genes, etc.

Cloning Services - Knockout Plasmid | Precise and High efficiency

CRISPR/Cas9 system is a defense mechanism used by bacteria to resist the invasion of viruses and foreign plasmids. At present, the type II CRISPR/Cas9 system is the most developed and widely used system. It 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 a DNA sequence via Homology Directed Repair (HDR) or Non-Homologous End Joining (NHEJ).

Ubigene developed a series of YKO plasmids, including lentivirus plasmids, AAV plasmids, and non-viral plasmids, which can be widely used in vitro or in vivo.

The plasmid can be used in frame-shift mutation or short fragment knockout. By co-transfecting 2-3 gRNAs, knockout efficiency can be improved.

Dual-gRNA plasmid can be used in large fragment removal. Dual-gRNA can greatly improve the efficiency of cell transfection and gene knockout.

CRISPR-B™ in Fungus and Bacterial | Precise and High efficiency

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.

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. 

CRISPR-U™ Knockdown stable Cell line | High efficiency

The knockdown vector would be transferred into the cells by lentivirus transduction. After drug screening, WT cells die out and a positive cell pool obtained.

CRISPR-U™ Overexpression stable Cell line | High efficiency

The overexpression vector would be transferred into the cells by lentivirus transduction or piggyBac transposition. After drug screening, WT cells die out and a positive cell pool obtained.

CRISPR-U™ Point mutation Cell Line

CRISPR/Cas9 and ssODN used to repair the point mutation in A79V-hiPSC. A) 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). 

CRISPR-U™ Knockin 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).

ACE2 CRISPR KO cell line, High Efficiency

ACE2 KO cell line

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 an 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 the 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 the 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 a 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 the 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) the 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 anesthetized 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, the 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.