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. 2020 Aug 7;10:13332. doi: 10.1038/s41598-020-70258-w

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licens»e, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Targeted deletions of Venus transgene by co-electroporation of two gRNA-encoding plasmids. (a) End-point PCR showed deletion of 15 different fragments from the original amplicon (1204 bp) using pairwise combination of gRNAs targeting the promoter/ 5′ region (− 72, − 69, + 36, + 100, and + 121) and the 3′ region (+ 518, + 554, and + 676) of Venus cDNA. The primer set (Venus-Forward1 and Venus-Reverse2) amplified both the shortened and original fragments. Genomic DNA from MEF cells carrying no-copy of Venus was considered as the negative controls (− Ve control). (b) Digital-PCR results for quantification of the rate of targeted deletion via a primer–probe assay. The primer–probe assay was designed to amplify only the wild type fragment. The deletion rate was calculated as the rate of copy number/µl in the co-electroporated groups divided by that of their control (non-treated) counterparts. Because of a poor DNA quality, the combined group of gRNA + 518 and + 100 was removed from the digital PCR analysis. Data are depicted as average ± standard error (n > 3).