Thursday, February 27, 2020

CRISPR gene editing technology: is more effective than RNAi and best choice for most biological research.

shRNA vs gRNA:
RNAi technology used to be popular in biological research, especially in high-throughput genetic screening, gene function study, clinical treatments for antivirus and cancers. However, the RNAi technology has been questioned because of its off-target effect. In recent years, the emerging CRISPR gene editing technology is widely used in almost every biological field. Because of its high specificity, low off-target effect and wide application prospects, CRISPR is replacing RNAi as the best choice for most research.

What RNAi might cost you?
One of the biggest limitations of the RNAi silencing method is that it suffers from High off-target effects. RNAi might silence unintended RNA targets resulting in modified phenotypes and therefore influence gene function screening experiments. There are three reasons for this high off-target effect:

· siRNA may compete with the intrinsic microRNA in RISC complex, resulting in affecting the function of microRNA and the expression regulation of other genes;
· siRNA also targeted sequences with limited complementarity;
· siRNA may interact with the 3'UTR of unintended RNA, leading to its degradation.

Other than high-off target effect, issues researchers are facing with are that:
· shRNA fails to show any reduction in protein expression level;
· No suppression but overexpression of target genes;
· Transcripts with high turnover are difficult to silence;
· Non-coding sequences are difficult to silence.
CRISPR has replaced RNAi as the best choice for most research applications:
Researchers conducted parallel experiments with shRNA libraries and CRISPR gRNA libraries, which proved that the efficiency and reliability of using gRNA libraries to screen genes was much higher than that of shRNA libraries.

In addition to high efficiency and high true positive rate, the off-target effect of CRISPR is very low. Because its specificity depends on two aspects, one is the base pairing between gRNA and target DNA, the other is that the Cas9-gRNA complex only binds to the PAM sequence in the genome. Only when these two conditions are met at the same time can Cas9 cause DSB.

Although RNAi was adopted as a gene silencing technique first, CRISPR has surpassed RNAi in popularity due to several advantages made possible by advancements that have refined CRISPR technology. The main reason for the popularity of CRISPR is it's specific yet versatile nature. At the same time, the choice remains with the user to perform knockouts, knock-ins, or knockdown experiments, making CRISPR extremely versatile.
Combine RNAi with CRISPR technology:
In some cases, CRISPR and RNAi can work with each other. For instance, it is very rare for any drug to achieve 100% inhibition of the target gene, so some scientists believe that a more rigorous and ideal experiment design is to use shRNA to validate a phenotype observed by CRISPR technology.

· Combined shRNA over CRISPR/cas9 as a methodology to detect of-target efects and a potential compensatory mechanism
· CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes
· Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes
· Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR

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Tuesday, February 25, 2020

THP-1 Knockout Cell Lines: an ideal tool for studying immunity and inflammation

CRISPR/Cas9 mediated THP-1 Disease Models

A human leukemic cell line (THP-1) cultured from the blood of a boy with acute monocytic leukemia. Since its establishment in 1980, THP-1 cells have been widely used in the research of monocyte and macrophage related mechanisms, signaling pathways, nutrient and drug transportation. The morphological and functional characteristics of THP-1 are very similar to human primary monocytes (including cell differentiation markers). Compared with human peripheral blood monocytes (PBMC), THP-1 is easier to be cultured and has a more consistent background. Therefore, THP-1 is a commonly used acute monocytic leukemia cell line in various laboratories, and it is an ideal tool for studying immunity and inflammation.

Application of THP-1: macrophage and inflammation model  
THP-1can be differentiated into M1/M2 macrophages and release corresponding cytokines.

M1 macrophage polarization:
  • THP-1 can be induced to differentiate into macrophages by Phorbol 12-myristate 13-acetate (PMA), and then M1 polarization can be induced by lipopolysaccharide (LPS) and IFN -γ, releasing TNF -α, IL-6 and other cytokines. This is a typical inflammatory model.

M2 macrophage polarization:
  • M2 polarization can be induced by IL-4, IL-13 and macrophage colony-stimulating factor (M-CSF). TGF - β, IL-10 and other inhibitory cytokines can be released. This is similar to the process of tissue repair and reconstruction in the late stage of inflammation.
Atherosclerotic inflammation model: Under the action of oxidized low-density lipoprotein (ox-LDL), macrophages can further become foam cells. This is a pathological cell in atherosclerotic plaques and is a chronic inflammation model.

The combination of THP-1 and CRISPR/Cas9 technology is helpful for the study of immune and inflammatory diseases  
THP-1 is a near-tetraploid suspension cell, and the success rate of THP-1 is very low by conventional gene-editing methods. CRISPR/Cas9 is widely used to construct gene-editing THP-1 model because of its simple, high efficiency and low toxicity.

Key genes for macrophagesclearing pathogens was found by CRISPR/Cas9 mediated gene knock-out THP-1 model   
 Phagosome acidification of macrophages is an essential step to eliminate pathogens. Phagosome acidification is closely related to the metabolism of macrophages and the transportation of nutrients. And the transportation of metabolites is closely related to solute carrier (SLC)protein. The researchers found that the bicarbonate transporter SLC4A7 in the SLC family is an essential gene for phagosome acidification of macrophages. In CRISPR/Cas9 mediated SLC4A7 knockout THP-1 cell line, the ability of phagosome acidification and killing bacteria was reduced. The acidity of the phagocyte was increased after the supplementation of SLC4A7. This indicates that SLC4A7 mediated bicarbonate driven in macrophages is essential for the maintenance of cytoplasmic pH and phagosome acidification. CRISPR-UTM can efficiently transfer gRNA and Cas9 into THP-1 cells by nucleofection. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

CRISPR/Cas9 mediated Chronic granulomatous disease (CGD) THP-1 cell line model is helpful to develop better disease treatments  
Chronic granulomatous disease (CGD) is a rare X-linked genetic disease. Due to the mutation or deficient of  CYBB gene, macrophages lack nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and cannot produce hydrogen peroxide to effectively kill the invading microorganisms. This usually leads to serious repeated infections caused by bacteria, fungi and other microorganisms. Some researchers used CRISPR/Cas9 technology to knockout  CYBB gene and generate point mutation c.90c>G (found in a CGD patient) in THP-1 cells, and successfully constructed the CGD model. Compared with wild-type THP-1 cells, two KO clones (#3 and #27) and a point mutation clone (#2, c.90c > G) showed decrease in H2O2 level after PMA and LPS induction, and a significant increase in IL-1β, TNF-α and IL-6 release, which was consistent with the behavior of macrophages in CGD. This CGD model provides a powerful tool for disease study and will help to develop better treatments.

By using CRISPR-UTM,THP-1 cell line would be efficiently co-transfected with gRNA, Cas9 and ssODN. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

The signal pathway of intracellular antiviral response was confirmed by the THP-1 Cell Models of gene knock-out and knock-in.  
The abnormal location of DNA in the cytoplasm is usually related to virus infection or tumor. The cGAS-cGAMP-STING pathway can detect the existence of cytosolic dsDNA, and induce a strong immune response, producing interferon and activating other immune response genes. RIG1-MAVS can detect pppRNA (dsRNA, the genome of some viruses) in cytoplasm and induce immune response. Sometimes there is a complex of RNA and DNA in the cytoplasm, which usually occurs in the case of some virus infection. In order to study which pathway that the RNA-DNA complex activates the immune response, the researchers generatedMAVS, cGAS, STING knockout THP-1 cell lines, and introduced dsDNA, ppRNA and RNA-DNA complex into the cells respectively. It was found that the RNA-DNA complex is activated by the cGAS-cGAMP-STING pathway.

Then, the researchers used CRISPR/Cas9 technology to insert2A-GLuc into the IFIT1 gene. IFIT1 is a typical interferon activated gene. Subsequent experiments showed that after the introduction of RNA-DNA complex, the expression of Gluc was driven by the activation of IFIT1 promoter due to the expression of interferon. These results further proved that the RNA-DNA complex in the cytoplasm activated the immune response through the cGAS-cGAMP-STING pathway. By using CRISPR-UTM,THP-1 cell line would be co-transfected with gRNA, Cas9 and donor vector. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

CRISPR-U™ efficiently modify genes in THP-1 cell line

CRISPR-U™, 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). Ubigene can customize the gene-editing THP-1 cell line and other monocytes that you are interested in. We can also help you to generate various genes overexpression in THP-1 cell line.

[1]Tsuchiya S, Yamabe M, Yamaguchi Y, et al. Establishment and characterization of a human acute monocytic leukemia cell line (THP‐1)[J]. International journal of cancer, 1980, 26(2): 171-176.  
[2] Chanput W, Mes J J, Wichers H J. THP-1 cell line: an in vitro cell model for immune modulation approach[J]. International immunopharmacology, 2014, 23(1): 37-45.  
[3] Sedlyarov V, Eichner R, Girardi E, et al. The bicarbonate transporter SLC4A7 plays a key role in macrophage phagosome acidification[J]. Cell host & microbe, 2018, 23(6): 766-774. e5.  
[4] Benyoucef A, Marchitto L, Touzot F. CRISPR gene-engineered CYBBko THP-1 cell lines highlight the crucial role of NADPH-induced reactive oxygen species for regulating inflammasome activation[J]. Journal of Allergy and Clinical Immunology, 2020.  
[5] Mankan A K, Schmidt T, Chauhan D, et al. Cytosolic RNA: DNA hybrids activate the cGAS–STING axis[J]. The EMBO journal, 2014, 33(24): 2937-2946.

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    Sunday, February 23, 2020

    Knockout Cell Lines: HEK293,Hela,HCT116,A549,CHO and more

    Blessing 2020! Your experiments, Ubigene cares

    2020 is a challenging year. Even though a lot of things are unexpected, our efficient and affordable genome-editing services are always excepted.

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    Versatility: Easily generate knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo.
    NO risk: CRISPR-U™ offers a 100% mutation guarantee. No mutation, no charge!

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