YOGESH CHANDER, RAM KUMAR, NITIN KHANDELWAL, ASSIM VERMA, KRISHAN DUTTA RAWAT, NAMITA SINGH, SANJAY BARUA* AND NAVEEN KUMAR*
National Centre for Veterinary Type Cultures, ICAR-National Research Centre on Equines, Hisar-125 001 (Haryana), India
*(e-mail: Naveen.Kumar1@icar.gov.in; Mobile: 99921 17990; and Sanjay Barua (sbarua.icar@gmail.com; Mobile:
98969 42824)
(Received : October 20, 2022; Accepted : November 24, 2022)
ABSTRACT
CRISPR/Cas9 technology has gained immense popularity in last decade due to its ability to knockout gene of interest in target cells. In the present study, recombinant pL.CRISPR.EFS.GFP vector having five pairs of gRNA designed against CBX5 gene was generated. When it was transfected in HeLa cell line, the vector successfully knocked out the target gene. The knockout was preliminary confirmed by PCR-based amplification of region of interest. The sangar sequencing revealed the precise deletion of 369 bp from CBX5 gene rendering it “shut down” completely. Despite the knockout of this gene, no significant changes in cell morphologies were observed when compared with WT HeLa cells which demonstrated the dispensable nature of the gene for HeLa cell line. Furthermore, the detailed methodology for knocking out the CBX5 gene has been provided.
Key words : CRISPR/Cas9, knockout, CBX5, gRNA, HeLa