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.
