Development of a CRISPR-based toolkit for the targeted Disruption of CD36 in human oral cancer cells

Abstract

CD36 (Cluster of Differentiation 36) is a transmembrane glycoprotein that plays a crucial role in various cellular processes, including lipid metabolism and cell adhesion, and has been implicated in cancer progression, metastasis, chemoresistance, and radio-resistance. The disruption of the CD36 gene may change the behaviour of the cancer cell, highlighting its potential as a therapeutic target in cancer treatment. CRISPR technology has revolutionised genome editing, enabling precise modifications of specific genes. This study aims to design a CRISPRbased toolkit for the effective disruption of CD36 in human cancer cells using the Homologydirected Repair (HDR) pathway. A total of 66 sgRNAs were designed using ATUM, Horizon’s CRISPR Design Tool, and CHOP CHOP Harvard, and three sgRNAs were selected based on the target location, on-target efficiency, and minimised off-target effects. The sgRNAs were selected to target early exons (exon 3 and exon 5) to enhance knockout efficiency, for the efficient production of a non-functional truncated protein. The selected sgRNAs exhibit 100% sequence similarity to all mRNA variants of CD36, with no predicted off-target effects. The designed sgRNAs were synthesised and cloned into the pSpCas9(BB)-2A-Puro vector, and the recombinant plasmids were verified through colony PCR and sequencing. Donor templates for HDR-mediated repair were designed to incorporate an EcoRI restriction site (GAATTC) and a 6xHis-tag (CATCACCATCACCATCAC-Stop) at the double-stranded break, which permits identification of the target PCR amplicon via restriction enzyme digestion and truncated proteins viaWestern blot. The sgRNA-cloned recombinant vectors, along with their corresponding donor templates, will be co-transfected into oral cancer cells in vitro to facilitate the knockout of CD36. Knockout will be confirmed through PCR, sequencing, RT-PCR, and Western blot analysis. This CRISPR knockout strategy provides a robust approach to elucidate the role of CD36 in tumor biology and possesses potential for the development of targeted therapy against CD36 for the treatment of cancer.