Engineering an analog-sensitive CDK12 cell line using CRISPR/Cas.

Abstract

The RNA Polymerase II C-terminal domain (CTD) kinase CDK12 has been implicated as a tumor suppressor and regulator of DNA damage response genes. Although much has been learned about CDK12 and its activity, due to the lack of a specific inhibitor and the complications posed by long term RNAi depletion, much is still unknown about the particulars of CDK12 function. Therefore gaining a better understanding of CDK12's roles at the molecular level will be challenging without the development of additional tools. In order to address these issues we have used the CRISPR/Cas gene engineering system to create a mammalian cell line in which the only functional copy of CDK12 is selectively inhibitable by a cell-permeable adenine analog (analog-sensitive CDK12). Inhibition of CDK12 results in a perturbation of the phosphorylation patterns on the CTD and an arrest in cellular proliferation. This cell line should serve as a powerful tool for future studies.

• Authors: Bartlomiej Bartkowiak, Christopher Yan, Arno L Greenleaf
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 1849 Issue 9 Pg. 1179-87 (Sep 2015) ISSN: 0006-3002 [Print] Netherlands
• PMID: 26189575 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
• Copyright: Copyright © 2015 Elsevier B.V. All rights reserved.

Chemical References

• DNA
• CDK12 protein, human
• Cyclin-Dependent Kinases

Topics

• Base Sequence
• Clustered Regularly Interspaced Short Palindromic Repeats (genetics)
• Cyclin-Dependent Kinases (genetics)
• DNA
• Gene Knockdown Techniques
• HeLa Cells
• Humans
• Molecular Sequence Data
• Sequence Homology, Nucleic Acid