circRNA was studied by CRISPR knockout cell lines.

circRNAs are circular noncoding RNAs formed by reverse splicing of pre-mRNAs. circRNAs were firstly found in viruses in the 1970s. However, due to the extensive use of the method for enrichment of poly (A) (no 5 'and 3' ends of circRNA) in the early RNA library preparation, and the calculation algorithm that RNA-seq reading requires linear alignment with genome, a large number of circRNA information was omitted, which led to the belief that circRNA is just a byproduct of miss splicing.

With the development of high-throughput sequencing technology and bioinformatics, thousands of circRNA have been found, and more and more basic researches related to circRNA have been done. A large number of studies have shown that circRNA is endogenetic, abundant, conservative and stable in mammalian cells, and often shows tissue or space-time specificity. It can participate in the regulation of cell growth and development, as well as the occurrence and development of diseases through a variety of mechanisms. Therefore, in recent years, circRNA has become popular in the field of non-coding RNA research.

Relationships between circRNAs and diseases

At present, the most studied is the relationship between circRNAs and tumors. Some circRNAs promote tumor formation, such as circPvt1 in squamous cell carcinomas of the head and neck, cirs-7 (CDr1as) in colorectal cancer, esophageal squamous cell carcinoma and hepatocellular carcinoma. Some circRNAs suppress tumors, such as circsMARCA5 and circ-SHPRH in glioblastoma. Some circRNAs may play different roles in different tissues or cells, such as circHiPK3, which is a proto-oncogene in rectal cancer, but suppresses cancer cells in bladder cancer.

In addition to cancers, circRNA has been found to be closely related to diabetes, cardiovascular disease, chronic inflammation and nervous system diseases. It is believed that with the development of biotechnology and more in-depth researches on circRNA, the formations and mechanisms of circRNAs can be identified. circRNAs can play important roles in disease prevention, diagnosis and treatment discovery.

The engineering and regulation of circRNA help disease studies and treatment discoveries

circRNA involves many complex functions, so how to study its functions? Similar to protein-coding genes, the most common methods are knockout, knockdown (RNA interference) or overexpression of the circRNA.

CRISPR/Cas9 mediated circRNA knockout, reveals the mechanism of its regulation on tumor formation

circRNA knockout refers to editing at the level of DNA to achieve the purpose of a complete knockout. The most commonly used method is to design two gRNAs at both ends of the circRNA exon to knockout the whole cyclized exon sequence. Although this strategy can knockout the circRNA, it will also affect the parent gene encoding the protein, and the study on its function is not ideal.

More and more researchers have given up using this strategy to knockout the circRNA because of its great influence on the parent gene. The ideal method is to knockout the loop forming elements (Alu) in the flanking intron of the exon, so as to destroy the circRNA loop forming without affecting the expression of the coding gene. However, it is difficult for many researchers to master the logic of designing the targeting strategy.

Taking circ-HIPK3 as an example, circ-HIPK3 is a kind of circRNA rich in human cells, which can combine with a variety of miRNAs as a regulator of cell growth and affect the formation of tumors. In order to verify how circ-HIPK3 forms into circle, it is necessary to find the loop forming elements in flanking intron. A pair of sgRNA is designed for the two Alu elements predicted at upstream and downstream respectively. The predicted loop forming elements are knockout by CRISPR/Cas9 system to detect whether the expression of circRNA changes. After PCR and RT-qPCR verification, it was found that the expression of circ-HIPK3 was significantly down-regulated after knockout of downstream loop forming elements, while the expression of circ-HIPK3 was not decreased but increased after knockout of upstream loop forming elements. It was speculated that there were too many loop forming elements in the upstream and the prediction was not accurate. In order to further verify the RNA circulation driven by other elements, the large fragment of intron in the upstream of the element was knockout by co-injection of gRNA3 or gRNA4 with gRNA5 or gRNA6. RT-qPCR results showed that the expression of circ-HIPK3 decreased, indicating that other loop forming elements of circ-HIPK3 exist.

Ubigene is experienced in designing strategy of knockout the loop forming elements in the flanking intron of circRNA exon, to achieve the purpose of knockout circRNA without affecting the expression of coding gene. Combined with CRISPR-UTM technology, the positive clones of circRNA knockout can be generated 10x faster than other common methods.

Knockdown specific circRNA and disclose its regulatory mechanism on cell proliferation and apoptosis

Among the methods to study the function of circRNA, the most classical way to inhibit circRNA is to knockdown it by RNAi (shRNA or siRNA). In order to avoid affecting mRNAs of coding genes, the shRNA should be designed at the back splicing site (BSS). siRNA was used to interfere with circ-HIPK3, and whether the knockdown of circ-HIPK3 would affect cell proliferation or apoptosis was observed. First, three groups of experiments targeting the linear transcript of HIPK3 mRNA, circ-HIPK3 circular transcript and both two transcripts. The designed siRNA interfered with the corresponding transcripts was verified on HEK-293T cell line. Cell proliferation and apoptosis were detected by CCK-8 and EdU assays. The results showed that the knockdown circ-HIPK3 significantly inhibited cell proliferation.

Ubigene can design high-score shRNA and use lentivirus to transfer the RNA interference vector into the cells. Cells were screened according to the drug screening, and the stable cell lines with circRNA knockdown were obtained.

Overexpression of circRNA reveal its loop-forming mechanism

Overexpression of circRNA is not easy because of its low efficiency of loop forming, and it is easy to mismatch. By optimizing the binding sites of RBP, such as Alu elements and QKI, the circRNA can be formed accurately and efficiently. After overexpression, it is necessary to detect successful loop-forming and linear mRNA expression. In order to study the loop-forming efficiency of a new circRNA expression system, the mouse circrtn4 gene was selected to express in a variety of cell lines (including HeLa, N2a, HEK293). According to the RT-qPCR results in different cell lines, the efficiency of the new vector system pCircRNA-DMo-Rtn4 is much higher than that of the common vector system (pCircRNA-BE-Rtn4) in these cell lines.

CRISPR-U™ high-efficiency gene editing system

Ubigene focuses on genome editing, CRISPR-U™ is a gene-editing technology developed by Ubigene, which is more efficient than common CRISPR/Cas9 technologies in gene targeting. The services related to circRNA editing introduced in this paper can be provided by Ubigene, including knockout, interference, overexpression and circRNA expression testing.

Check out our list of cell lines that we had successfully modified

References:
[1] Kristensen, L. S., Andersen, M. S., Stagsted, L. V., Ebbesen, K. K., Hansen, T. B., & Kjems, J. (2019). The biogenesis, biology and characterization of circular RNAs. Nature Reviews Genetics, 20(11), 675-691.
[2] Santer, L., Bär, C., & Thum, T. (2019). Circular RNAs, a novel class of functional RNA molecules with therapeutic perspective. Molecular Therapy.
[3] Zheng, Q., Bao, C., Guo, W., Li, S., Chen, J., Chen, B., ... & Liang, L. (2016). Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nature communications, 7(1), 1-13.

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The combination of iPSC and CRISPR/Cas9 opens a new window for diseases research

Many serious diseases cannot be cured by medicines, such as heart failure, Late Stage Diabetes, hemophilia, myeloma, End-Stage Cirrhosis, etc. The best method is allogeneic transplantation. However, due to the limited donors and the risk of immune rejection, researchers are dedicated to finding more efficient and safer treatment besides allogeneic transplantation. Induced pluripotent stem cells (iPSCs) can be derived from the body cells of the patients themselves, which eliminates the risk of immune rejection, and has the potential of differentiation into different cells. Transplantation of cells derived from iPSC, such as cardiomyocytes, hepatocytes, neurocytes, T cells, hematopoietic stem cells (HSCs) and islet cells, is possible to solve many medical problems. By CRISPR/Cas9 technology, the mutations that simulating diseases could be introduced into iPSC. Using CRISPR/Cas9 to repair the mutations in iPSC disease models is also a popular application.

CRISPR/Cas9 gene editing in iPSC opens a new window for diseases research

The success rate of gene editing in human iPSC is lower because, unlike tumor cell lines, iPSC does not have the characteristics of chromosomal abnormality and strong ability of DNA repair. CRISPR/Cas9 has the advantages of high efficiency, easy to construct and low toxicity in human cells, so it is the most common method in iPSC genome editing.

CRISPR/Cas9 mediated RAG2 gene knockout in iPSC, generates CD8αβ - T cells with stable antigen specificity

The limited T cells and the difficulty of proliferation is the main obstacle of T-cell immunotherapy, which can be overcome by using pluripotent stem cells with proliferation and differentiation ability to generate T-iPSC with antigen specificity. Strict antigen specificity is essential for safe and effective T-cell immunotherapy. However, in the process of double-positive CD4/CD8differentiation, the rearrangement of the T-cell receptor (TCR) α chain will lose antigen specificity. This TCR rearrangement was prevented by removing the recombinant enzyme gene (RAG2) in T-iPSCs with CRISPR/Cas9.  Xenotransplantation of CD8αβ-T cells with stable TCR can effectively inhibit tumor growth in disease models. This contributes to a safe and effective T-cell immunotherapy.（Minagawa, Atsutaka, et al.）

CRISPR-U^TMcan efficiently transfer gRNA and Cas9 into iPSC by nucleofection. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

CRISPR/Cas9 was used to repair the point mutation of iPSC disease model derived from an AD patient' cells

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease, which can lead to degeneration of nerve cells and atrophy of brain. It is considered as the most common form of dementia. The A79V mutation of PSEN1 gene can cause Alzheimer's disease. By studying the effect of this mutation on cell phenotype, researchers can further study the pathology of this disease and develop a more effective treatment. The researchers reprogrammed the somatic cells of a patient into pluripotent stem cells (iPSCs), and then replaced the mutated gene with a wild-type sequence. By studying the disease model and the modified iPSC, the effect of the mutation on cell phenotype can be determined, so as to further study the pathological effect of the mutation.（Pires, C., et al.）

With CRISPR-U^TM, iPSCwould be co-transfected with gRNA, Cas9 and donor oligo by electroporation. After the DNA DSB caused by the complex of gRNA and Cas9, iPSCs use donor oligo carrying wild-type sequence as a template for homologous recombination repair (HDR) and replace the target sequence with point mutation.

Hemophilia B can be treated by iPSC differentiated hepatocytes with AAVS1 safe harborknockin Coagulation factor IX (F9)

The most common method to treat hemophilia is substitution therapy, but this method has the risk of virus infection, and it is a method that needs lifelong continuous treatment. Gene therapy seems like the only way can cure hemophilia. CRISPR/Cas9 technology can be used for gene therapy of hemophilia. The mutations of coagulation factors, F8 and F9, are the main causes of hemophilia. Previous studies have shown that F9 is a more effective gene therapy target. AAVS1-Cas9-sgRNAplasmid and AAVS1-EF1α-F9 cDNA puromycin donor plasmid were constructed and transferred into iPSC. Human factor IX (hFIX) antigen activity was detected in the culture supernatant. Finally, liver cells differentiated from iPSC were transplanted into NOD/SCIDmice by spleen injection, to cure hemophilia B.（Lyu, Cuicui, et al.）

With CRISPR-U^TM, iPSCwould be co-transfected with gRNA, Cas9 and donor vector by electroporation. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

iPSC induced differentiation, making "autotransplantation" possible

The study of human embryonic stem cells (hESCs) derived from early embryos has been controversial in ethics, and the rejection of differentiated cells derived from hESCs intransplantation has limited its clinical application. Hepatocytes, nerve cells, T cells, cardiomyocytes, hematopoietic stem cells and pancreatic cells can be differentiated from patients' somatic cells (such as fibroblasts) or existing iPSCs.

iPSC Differentiation Process

iPSC reprogramming: Ubigenehas optimized the reprogramming method ——>iPSC induced differentiation: continuous addition of inducing factor in iPSC medium——>Cell validation: genotype, phenotype and cell characteristic analysis

Ubigene's iPSC platform

Ubigene focuses on the optimization of iPSC reprogramming, gene editing and differentiation, and has established a set of mature experimental procedures. With CRIPSR-U^TM technology, gene editing in iPSC is much accessible.

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