Cellectis - bioresearch

Targeting genes in endogenous loci

Meganuclease engineering allows us to target natural endogenous loci.

Depending on the locus that is targeted, the associated genetic elements and the selection strategy differ from one cGPS^® Custom product to another.

The first ever engineered meganuclease for the hamster genome targets the natural HPRT (Hypoxanthine-guanine PhosphoRibosylTransferase) locus. HPRT is involved in purine metabolism and converts 6-thioguanine (6-TG) to its monophosphate form (6-TGMP). 6-TGMP interferes subsequently with de novo synthesis of purines.

CHO-K1 HPRT+ cells are naturally sensitive to 6’TG. Upon expression of the engineered meganuclease targeting HPRT following cell transfection, a DNA double-strand break is made which triggers repair through homologous recombination. The transfected integration matrix is used for repair and contains upstream and downstream homology regions of the HPRT locus, a hygromycin resistance gene, and promoter and terminator sequences of the Gene Of Interest (GOI) coding sequence. Upon repair, the GOI is integrated at the HPRT meganuclease recognition site, and the cells become hygromycin and 6-TG resistant (HPRT deficient).