Correction of a CD55 mutation to quantify the efficiency of targeted knock-in via flow cytometry.

BACKGROUND: Targeted knock-in assisted by the CRISPR/Cas9 system is an advanced technology with promising applications in various research fields including medical and agricultural sciences. However, improvements in the efficiency, precision, and specificity of targeted knock-in are prerequisites to facilitate the practical application of this technology. To improve the efficiency of targeted knock-in, it is necessary to have a molecular system that allows sensitive monitoring of targeted knock-in events with simple procedures. METHODS AND RESULTS: We developed an assay, named CD55 correction assay, with which to monitor CD55 gene correction accomplished by targeted knock-in. To create the reporter clones used in this assay, we initially introduced a 7.7-kb heterozygous deletion covering CD55 exons 2-5, and then incorporated a truncating mutation within exon 4 of the remaining CD55 allele in human cell lines. The resultant reporter clones that lost the CD55 protein on the cell membrane were next transfected with Cas9 constructs along with a donor plasmid carrying wild-type CD55 exon 4. The cells were subsequently stained with fluorescence-labeled CD55 antibody and analyzed by flow cytometry to detect CD55-positive cells. These procedures allow high-throughput, quantitative detection of targeted gene correction events occurring in an endogenous human gene. CONCLUSIONS: The current study demonstrated the utility of the CD55 correction assay to sensitively quantify the efficiency of targeted knock-in. When used with the PIGA correction assay, the CD55 correction assay will help accurately determine the efficiency of targeted knock-in, precluding possible experimental biases caused by cell line-specific and locus-specific factors.