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

CRISPR J. 2020 Oct;3(5):409-418. doi: 10.1089/crispr.2020.0037.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Validation Study

MeSH terms

  • Animals
  • Animals, Genetically Modified / genetics*
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism*
  • Cells, Cultured
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • 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*

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • Trans-Activators
  • CRISPR-Associated Protein 9