Conditional knockout cell line is a technique used to eliminate specific genes in certain tissues, such as the liver. This technique is very useful for studying the role of individual genes in living organisms. It is different from traditional gene knockout technology because it targets a specific gene at a specific time instead of deleting it from the beginning. The use of conditional gene knockout technology eliminates many side effects of traditional gene knockout. In traditional gene knockout, embryos may die due to genetic mutations, which prevents scientists from studying adult genes. Certain tissues cannot be properly studied alone, so the gene must be inactive in some tissues, while it must remain active in other tissues. Using this technology, scientists can knock out genes at specific stages of development and study how knocking out genes in one tissue affects the same genes in other tissues.
Easi-CRISPR is used to create knock-in and conditional knockout mouse models using long ssDNA donors
CRISPR/Cas9-based genome editing can easily produce knockout mouse primary cells by disrupting gene sequences, but the efficiency of creating models that require insertion of foreign DNA (knock-in) or replacement of genome fragments is very low. Most of the mouse models used in this study involve knock-in (reporter gene or recombinase) or gene replacement (for example, conditional knockout alleles containing exons flanked by LoxP sites). Some methods of creating this model have been reported. These methods use double-stranded DNA as a donor, but the efficiency is usually 1-10%, so they are not suitable for routine use. Researchers have recently demonstrated that long single-stranded DNA (ssDNA) can be a very effective donor, whether for insertion or gene replacement. Researchers call this method an effective complement to ssDNA insert-CRISPR (Easi-CRISPR) because it is an effective technique (efficiency is usually 30-60%, and in some cases up to 100%). The agreement will take approximately 2 months to produce founder mice.
Conditional gene knockout produced by engineered CRISPR-Cas9 endonuclease reveals the role of coronary heart disease in the neural development of Caenorhabditis elegans
Conditional knockout animals are valuable tools for studying the basic mechanisms of cell and developmental biology. The researchers developed a conditional knockout strategy by manipulating the expression of the RNA-directed DNA endonuclease CRISPR-Cas9 in the somatic cell lineage of the nematode Caenorhabditis elegans. The researchers showed that this CRISPR-Cas9 somatic cell technology provides a fast and effective method to generate conditional gene knockouts in various cell types at different developmental stages. In addition, the researchers proved that this method is superior to our recently developed somatic TALEN technology and can generate multiple conditional knockouts in one step. By combining these technologies with live cell imaging, the researchers showed that the essential embryonic gene Coronin, which is related to abnormal human neurobehavioral dysfunction, can regulate the actin organization of central nematodes during neuroblast migration and nerve formation. And cell morphology. The researchers proposed that the somatic CRISPR-Cas9 platform is particularly suitable for biomedical research based on conditional gene editing.
Systematic analysis of human telomere dysfunction using inducible telomere/masking protein CRISPR/Cas9 knockout cells
CRISPR/Cas9 technology can use somatic cells to perform effective loss-of-function analysis of human genes. However, the study of essential genes requires conditional knockout (KO) cells. The researchers described the telomere/shelterin complex that can induce the production of CRISPR KO human cell lines. The subunits of TRF1, TRF2, RAP1, TIN2, TPP1 and POT1 interact with telomeres or can interact with other telomeres. Homozygous inactivation of several subunits is fatal in mice. Most studies on the loss of human telomere regulator function rely on RNA interference-mediated gene knockout, which has its own limitations. Our inducible CRISPR method allows us to more conveniently obtain a large number of KO cells, in which the necessary telomere regulators have been inactivated for biochemical and molecular research. Our systematic analysis revealed the functional differences between human and mouse telomere proteins in DNA damage response, telomere length and metabolic control, thus providing new insights for maintaining telomeres.
Ubigene's goal is to simplify genome editing. Ubigene developed CRISPR-U™ (based on CRISPR/Cas9 technology), which is more effective than ordinary CRISPR/Cas9 in double-strand breaks, and CRISPR-U™ can greatly improve the efficiency of homologous recombination and easily achieve knockout (in vivo, in vitro) Point mutation (PM) and knock-in (KI). With CRISPR-U™, Ubigene has successfully edited genes on more than 100 cell lines.
Miura H, Quadros R M, Gurumurthy C B, et al. Easi -CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors[J]. Nature Protocols, 2018, 13(1): 195-215.
Shen Z, Zhang X, Chai Y, et al. Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development.[J]. Developmental Cell, 2014, 30(5): 625-636.
Kim H, Li F, He Q, et al. Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells[J]. Cell discovery, 2017, 3(1).
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.