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. 2021 Feb;63(2):140-149.
doi: 10.1007/s12033-020-00290-8. Epub 2021 Jan 2.

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

Dong-Hwan Kim  1 , Joonbum Lee  1   2 , Yeunsu Suh  1 , Kichoon Lee  3   4
Affiliations

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

Dong-Hwan Kim et al. Mol Biotechnol. 2021 Feb.

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.

Keywords: CRISPR/Cas9; Gene editing; Guide RNA; Quail cells; Sequencing; Validation.

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