Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway
نویسندگان
چکیده
Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.
منابع مشابه
Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair
CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficien...
متن کاملEnhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery
The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific doubl...
متن کاملHighly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.
Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR...
متن کاملOptimization of DNA, RNA and RNP Delivery for Efficient Mammalian Cell Engineering Optimization of DNA, RNA and RNP Delivery for Efficient Mammalian Cell Engineering using CRISPR/Cas9
The CRISPR/Cas9 genome-editing platform is a versatile and powerful technology to efficiently create genetically engineered living cells and organisms. This system requires a complex of Cas9 endonuclease protein with a gene-targeting guide RNA (gRNA) to introduce double-strand DNA breaks (DSBs) at specific locations in the genome. The cell then repairs the resulting DSBs using either homology-d...
متن کاملA stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair.
The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) after aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway or, alternatively, for enhancing homology...
متن کامل