Gene knock-in is a technology that introduces or replaces exogenous functional genes into a specific genome site and obtains stable cell expression. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene knock-in activates the repair mechanism by breaking the DNA double-strand, mainly non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Although homology-directed repair has a low probability of occurring under natural circumstances, in the presence of an exogenous DNA template, donor DNA or concurrently cleaved donor DNA is inserted into the double-strand DNA break (DSB) site through HDR or non-HDR mechanisms, then mediates the precise knock-in of the exogenous gene. Glycogene knock-in has a wide range of applications in the research fields of gene function, disease modeling, drug screening, etc. It aids the study of glycosylation expression mechanisms and allows the development of glycomics to go further.
Fig.1 The strategy for establishing the knock-in cells. (Kentaro, et al., 2016)
At CD BioGlyco, our technologies for glycogene knock-in include efficient and scalable epitope labeling using Cas9 ribonucleoproteins and CRISPR/Cas9-mediated knock-in in cell lines. Our services include designing single guide RNA (sgRNA) according to the target gene sequence, and sgRNA cloned into the desired vector. Then, we recombinant plasmid and homologous recombination repair template transfection into the target cell, resistance screening. Lastly, combine with cell genomic polymerase chain reaction (PCR) detection and sequencing to determine whether the gene insertion is correct. Detect the expression and function of glycoprotein through a variety of biological methods. Based on the CRISPR/Cas9 system, we have developed HDR, single-strand annealing (SSA), NHEJ, microhomology-mediated end joining (MMEJ) or homology-mediated end joining (HMEJ) methods, combining these experimental schemes to customize the glycogen knock-in protocol for you. At the same time, we also provide different Technologies for Glycogene Editing and Glycogene Engineering Services, comprehensively helping your development in glycogenomics.
Fig.2 Knock-in cell line process. (CD BioGlyco)
CD BioGlyco has always adhered to the client-centric principle and developed a set of Strategies for Glycogene Editing. Moreover, we also provide precise Glycogene Knockout, Knockdown, Tagging, and Overexpression Services. We have already many experiences and won unanimous praise from clients. If you are interested in our service, please feel free to contact us.
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