Reporter for Nucleases
Surrogate reporter system
Targeting efficiency of Engineered nucleases ranges 1%~30% for the most cases. Although this efficiency is quite remarkable compared to conventional gene targeting techniques by homologous recombination, steps to isolate cells bearing desired mutations still require extensive screening.
Our knockout cell enrichment system has been developed to overcome such limitations. The use of surrogate reporters allows selective enrichment of cells that have acquired EN mediated mutations at target sites.
The surrogate reporters are composed of genes encoding two fluorescent proteins (red and green) linked by the EN targeting sequence. In the absence of EN expression, they are designed to express the red but not the green fluorescent protein as it is placed out of frame. Upon expression of ENs, which can induce double strand break at the target site in front of GFP, leading to frameshift mutations and expression of GFP. The expression of green GFP is strictly dependent on the presence of nuclease activity from ENs that specifically recognize the target sequence in the reporter construct.
Our surrogate reporter system is a wonderful tool to visualize and enrich the cells with gene editing event. Using surrogate reporter system, the frequency of finding the knock out cells can be improved by 3~20 fold.
Surrogate Reporter Systems
Cas9 co-transfection/selection vectors
Surrogate reporter vector with GFP for FACS
Surrogate reporter vector with GFP and H2KK for MACS
Surrogate reporter vector with GFP and Hygromycin-R
Cas9 co-transfection vector (CMV promoter)
Cas9, Hygromycin R, and GFP expression vector (CMV promoter)
Cas9, RFP, puromycin R expression vector (CMV promoter)
Cas9 co-expression vector (EF1a promoter)
Cas9, Hygromycin R, and GFP expression vector (EF1a promoter)
Cas9, RFP, and Puromycin R expression vector (EF1a promoter)