The CRISPR-Cas system is an adaptive immune system found in bacteria which helps protect against the invasion of other microorganisms. This system induces double stranded breaks at precise genomic loci (1) in which repairs are initiated and insertions of a target are completed in the process. This mechanism can be used in eukaryotic cells in combination with sgRNAs (1) as a tool for genome editing. By using this CRISPR-Cas system, in addition to the “safe harbor locus,” ROSAβ26, the incorporation of a target gene into a site that is not susceptible to gene silencing effects can be achieved through few simple steps. PCR amplification of the target genes , ROSA26 and mKate2, with a sgRNA scaffold and T7 promoter followed by in vitro transcription aim to produce an RNA product. This sgRNA product can be run through a digestion with Cas9 to validate cleavage of the genomic ROSA DNA template or mKate plasmid. Osteoblast mouse cells are transfected and labeled through partial uptake by the CRISPR mechanism, by cutting in the ROSA loci and repairing with pieces of the fluorescent mKate2 plasmid. These cells were measured via flow cytometry to give a percentage of red cells. This data shows the scaffolding construct created is targeted by the Cas9 endonuclease and through homologous repair the cells will incorporate the mKate2 target gene in vitro in MC3T3 mouse cells
