Abstract:
Clustered Regularly Interspaced Short Palindromic Repeats associated RNA guided endonuclease 9 (CRISPR-Cas9) system is a programmable genome engineering technique, which can be employed to facilitate efficient genome engineering by simply specifying a 20-nt targeting sequence (crRNA) which is complementary to the desired target site. This study utilizes CRISPR/Cas9 system to modify two vital genes, pfk1 and PFKFB3, which encodes PFK1 (Phosphofructokinase-1) and PFK2 (Phosphofructokinase-2) enzymes that are involved in regulation of glycolysis, cell cycle and apoptosis. To investigate the impact of PFK1 and PFK2 encoding gene knockout on cancer, MCF-7 cells were utilized. Specific target sites to achieve knockout of respective genes were discovered through bioinformatics tools followed by designing target specific crRNAs and repair donor template with desired properties for downstream assays. Then crRNAs specific for pfk1 and PFKFB3 were separately cloned into pSpCas9(BB)-2A-Puro plasmids. Verification of recombinant plasmids was achieved through colony PCR and sequencing. Sequencing results indicated that the crRNA sequence is integrated accurately to the intended site of the plasmid in the correct orientation. After the initial Polyethylenimine mediated transfection of recombinant plasmids carrying crRNA and Cas9 gene into MCF-7 cells, transformed cells were selected using Puromycin. Subsequent Western blot did not produce expected bands with desired intensity which was carried out to verify gene silencing. Future work includes optimization of transfection, downstream verification procedures such as T7 endonuclease assay and restriction digestion assay to validate CRISPR mediated knockout of pfk1 and PFKB3 genes and determining the effect of gene knockout on cancer cell growth and proliferation.
