In Vitro Validation of Transgene Expression in Gene-Edited Pigs Using CRISPR Transcriptional Activators

• Published in: CRISPR journal Vol. 3; no. 5; p. 409
• Main Authors: Polkoff, Kathryn M, Chung, Jaewook, Simpson, Sean G, Gleason, Katherine, Piedrahita, Jorge A
• Format: Journal Article
• Language: English
• Published: United States 01.10.2020
• Subjects: Animals; Animals, Genetically Modified - genetics; Cells, Cultured; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; Gene Editing - methods; Gene Expression; Nuclear Transfer Techniques; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Swine; Trans-Activators - genetics; Trans-Activators - metabolism; Transgenes
• ISSN: 2573-1602
• DOI: 10.1089/crispr.2020.0037

The use of CRISPR-Cas and RNA-guided endonucleases has drastically changed research strategies for understanding and exploiting gene function, particularly for the generation of gene-edited animal models. This has resulted in an explosion in the number of gene-edited species, including highly biomedically relevant pig models. However, even with error-free DNA insertion or deletion, edited genes are occasionally not expressed and/or translated as expected. Therefore, there is a need to validate the expression outcomes gene modifications before investing in the costly generation of a gene-edited animal. Unfortunately, many gene targets are tissue specific and/or not expressed in cultured primary cells, making validation difficult without generating an animal. In this study, using pigs as a proof of concept, we show that CRISPR-dCas9 transcriptional activators can be used to validate functional transgene insertion in nonexpressing easily cultured cells such as fibroblasts. This is a tool that can be used across disciplines and animal species to save time and resources by verifying expected outcomes of gene edits before generating live animals.