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A Novel White-to-Blue Colony Formation Assay to Select for Optimized sgRNAs

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Abstract

CRISPR/Cas9-mediated genome editing technology consists of a single-guide RNA (sgRNA), and the Cas9 endonuclease has the potential to treat genetic diseases in most tissues and organisms. In this system, the Cas9 protein can be directed to target genomic DNA sequences as “molecular scissors” with the guidance of sgRNAs. However, the target-specific activities of different sgRNAs are highly variable; thus, it is crucial to search for a simple, quick and economical method to screen for optimized sgRNAs with high target specificity. We have adopted and verified a newly developed white-to-blue colony formation assay to quickly screen for sgRNAs optimized for the EphA2 gene, which is highly expressed in hormone-resistant prostate cancer (PC-3) cells. This assay promises to screen for optimized sgRNAs more simply, rapidly, and efficiently. Our results suggest that the white-to-blue colony formation assay might be a useful screening strategy to quickly select for optimized sgRNAs.

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Acknowledgements

The authors were financially supported by the National Natural Science Foundation of China (Grant No. 81801754), the Second Affiliated Hospital of Soochow University Preponderant Clinic Discipline Group project funding (Grant No. XKQ2015009), Jiangsu Province Youth medical talent support program (Grant No. QNRC2016868), the Suzhou Science and Technology Planning Project (Grant No. SYS201727). In addition, Professor Li Kai has provided a great help to this supplementary experiment and the modification of the article. We were appreciated for his help very much.

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  1. Chaogang Wei and Tong Chen have contributed equally to the work.

  2. Li Xiao and Junkang Shen are co-corresponding authors.

Authors and Affiliations

  1. Department of Radiology, The Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, Suzhou, 215004, Jiangsu, People’s Republic of China

    Chaogang Wei, Tong Chen, Yueyue Zhang, Yanfeng Wang, Dai Shi, Zhen Jiang & Junkang Shen

  2. Center of Laboratory, The Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, Suzhou, 215004, Jiangsu, People’s Republic of China

    Kai Li & Li Xiao

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  1. Chaogang Wei
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Correspondence to Li Xiao or Junkang Shen.

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Wei, C., Chen, T., Zhang, Y. et al. A Novel White-to-Blue Colony Formation Assay to Select for Optimized sgRNAs. Mol Biotechnol 63, 1–12 (2021). https://doi.org/10.1007/s12033-020-00280-w

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  • DOI: https://doi.org/10.1007/s12033-020-00280-w

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