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Necessity for Validation of Effectiveness of Selected Guide RNA In Silico for Application of CRISPR/Cas9

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Abstract

Selection of guide RNA (gRNA) is important to increase the efficiency of gene editing in the CRISPR/Cas9 system. Due to the variation in actual efficiency of insertion/deletion (indel) mutation among selected gRNAs in silico, reliable methods for validation of efficiency of gRNA need to be developed. Three gRNAs with high on-target scores (72.0 for target 1, 65.4 for target 2, and 62.9 for target 3) were designed to target the quail retinol binding protein 7 (qRbp7) gene, and indel efficiencies were predicted by the Sanger sequencing and Inference of CRISPR Edits (ICE) analysis of sorted cell populations receiving the CRISPR/Cas9 vector. Unlike the order of on-target scores among 3 gRNAs, predicted rates of indel mutations were highest in gRNA2, intermediate in gRNA1, and lowest in gRNA3. This was confirmed by actual indel mutation rates, 51.8% in gRNA2, 31% in gRNA1, and 12.9% in gRNA3, which were calculated by sequencing individual allele cloned into a vector. These data showed a rapid and reliable method for estimation of the efficiency of selected gRNAs, providing a critical necessary step for successful gene editing for further applications.

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Funding

This research was funded by the United States Department of Agriculture National Institute of Food and Agriculture Grant (Award No. 2020-67015-31537).

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Authors and Affiliations

  1. Department of Animal Sciences, The Ohio State University, Columbus, OH, 43210, USA

    Dong-Hwan Kim, Joonbum Lee, Yeunsu Suh & Kichoon Lee

  2. Interdisciplinary Ph.D. Program in Nutrition, The Ohio State University, Columbus, OH, 43210, USA

    Joonbum Lee & Kichoon Lee

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  1. Dong-Hwan Kim
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  2. Joonbum Lee
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  4. Kichoon Lee
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by D-HK, JL, and YS. The first draft of the manuscript was written by D-HK, JL, and KL and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Kichoon Lee.

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Kim, DH., Lee, J., Suh, Y. et al. Necessity for Validation of Effectiveness of Selected Guide RNA In Silico for Application of CRISPR/Cas9. Mol Biotechnol 63, 140–149 (2021). https://doi.org/10.1007/s12033-020-00290-8

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  • DOI: https://doi.org/10.1007/s12033-020-00290-8

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