Abstract
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.
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Data availability
Data and materials will be available from the corresponding author upon reasonable request.
Code availability
Not applicable.
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Acknowledgements
We would like to thank Mr. Makoto Naruse and colleagues at the Institute of Comprehensive Medical Research, Division of Advanced Research Promotion at Aichi Medical University, for providing technical assistance, and Dr. Feng Zhang (Broad Institute) for providing the plasmids PX458, PX459, and PX462 via Addgene.
Funding
This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS; 18K14703 to TH, 19K09292 to SK, 18K08342 and 21K08426 to AO, 18H02645 to ST, 19K08668 to YH, and 17K07263 and 20K06613 to HK), the Nitto Foundation (to TH), Takeda Science Foundation (to TH and HK, respectively), Hirose International Scholarship Foundation (to SK), Ichihara International scholarship foundation (to HK), and Takahashi Industrial and Economic Research Foundation (to HK). MLR and MNH are supported by the Japanese Government (MEXT) Scholarship for Research Students.
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HK conceived, designed, and supervised the project. HK designed and MLR performed the experiments. TH guided MLR in performing experiments. HK, TH, and MLR analyzed data. MNH, YM, SK, AO, ST, and YH provided insights into the study. HK wrote the manuscript with contribution from MLR.
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Rahman, M.L., Hyodo, T., Hasan, M.N. et al. Correction of a CD55 mutation to quantify the efficiency of targeted knock-in via flow cytometry. Mol Biol Rep 49, 6241–6248 (2022). https://doi.org/10.1007/s11033-022-07422-0
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DOI: https://doi.org/10.1007/s11033-022-07422-0