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

ABSTRACT: 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 in vitro 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.

SUBMITTER: Polkoff KM 

PROVIDER: S-EPMC7580606 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature