Abstract: Transforming growth factor-β activated kinase1 (TAK1) is a serine threonine kinase stimulated by TGF- β, in addition to other stimuli including inflammatory cytokines, microbial products and environmental stress. TAK1 regulates multiple cellular events notably developmental processes, differentiation, autophagy, apoptosis and cell survival through extensive cross-talk with crucial signalling pathways like, MAP kinases, IKK and JNK. On one hand it engages with energy sensor mechanisms via AMPK and on the other with growth regulation processes dominated by mTOR signaling. By creating a stable TAK1 knockout cell line, we can dissect how loss of TAK1 affects mTORC1 activation, autophagy induction, and cell survival under conditions of nutrient restriction or metabolic stress, thereby clarifying whether TAK1 functions as a pro‑survival, pro‑death, or context‑dependent modulator in hyperproliferative and aging‑like states. For this, three sgRNAs were designed for TAK1 gene. The sgRNA1 and sgRNA2 with the higher activity were respectively used to construct pSpCas9(BB)-2A-Puro-sgRNA1 and pSpCas9(BB)-2A-Puro-sgRNA2. The knockout plasmid vectors were transfected into HEK-293 cells. Through drug screening, the TAK1 gene-knockout HEK293 (TAK1-/-) cell lines was obtained which was subsequently confirmed with Western blotting and sequencing. This isogenic CRISPR‑derived knockout line will also provide a clean genetic background for rescue experiments and pharmacological studies, improving mechanistic rigour and enabling us to identify TAK1‑dependent nodes that could be targeted for therapeutic intervention in cancer or age‑related pathologies.
Keywords: CRISPR/Cas, TAK1, mTOR, Puromycin.
Title: Targeted disruption of TAK1 in HEK293 cells via CRISPR/Cas 9: A tool for functional dissection of inflammatory and autophagic signaling
Author: Tawseeq Abdullah, Waseem Rashid, Asiya Batool, Mohammad Afzal Zargar, Khurshid Iqbal Andrabi
International Journal of Life Sciences Research
ISSN 2348-313X (Print), ISSN 2348-3148 (online)
Vol. 14, Issue 2, April 2026 - June 2026
Page No: 5-11
Research Publish Journals
Website: www.researchpublish.com
Published Date: 01-May-2026
