Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) can be concocted to cleave any desired sequence in the genome. We combine its precision with the efficiency of flippase based homologous recombination (HR) to insert genes at a safe genomic locus like Adeno-associated virus integration site 1 (AAVS1) in seven different cell lines. Our approach involves the creation of an acceptor cell line tagged with flippase recognition target (FRT), which accelerates the generation of the desired transgenic cell line with a single-copy insertion at a specific locus. To create this acceptor cell line, we designed a series of guide RNAs (gRNAs) that recognize and cleave a specific sequence within the AAVS1 locus. These validated gRNAs were co-expressed with an FRT-tagging plasmid, which expresses reporter genes (fluorescence and/or antibiotic resistance) flanked by FRT-recombination and genomic sequences adjacent to the CRISPR site. This process resulted in the generation of an acceptor cell line tagged with FRT and reporter genes, using CRISPR-induced HR. Subsequently, a donor plasmid containing mCherry (our gene of interest/GOI) and reporter genes flanked by FRT was introduced into the acceptor cell line. This led to the insertion of the GOI and reporter genes in the tagged region at a neutral locus of the genome. Our strategy helps generate transgenic cell lines with precision, thereby offering a streamlined and efficient approach to genetic manipulation.

Key words: CRIPR-Cas9, guide RNA (gRNA), RMCE (FLP), AAVS1, Homologous Recombination.