In silico characterization and analysis of transcriptional inhibition of EGFR in childhood brain stem glioma tumor cells by CRISPR-C13a technique

A brainstem glioma is a cancerous glioma tumor in the brainstem. Around 75% are diagnosed in children andyoung adults under the age of twenty, but have been known to affect older adults as well. Brainstem gliomas startin the brain or spinal cord tissue and typically spread throughout the nervous system. The epidermal growthfactor receptor (EGFR) is a transmembrane protein that is a receptor for members of the epidermal growthfactor family (EGF family) of extracellular protein ligands. Mutations that lead to EGFR overexpression (knownas upregulation)/amplification have been associated with a number of cancers, including glioblastoma (50%)and epithelian tumors of the head and neck (80-100%). These somatic mutations involving EGFR lead to itsconstant activation, which produces uncontrolled cell division. In glioblastoma a more or less specific mutationof EGFR, called EGFRvIII is often observed. RNA targeting was a challenging issue in genetic engineering.Even CRISPR-Cas with its great applications, cannot target RNA. Recently, researchers have found out that aC13a protein in Leptotrichia shahii bacteria has two RNase activities, one for cutting its RNA target and theother for processing the CRISPR RNA (crRNA). Functional studies revealed that C13a is a RNA-guided RNAtargetingCRISPR effector. We design crRNA by CRISPR-RT for targeting EGFR RNA. After designing crRNAthat targets the EGFR RNA, its specificity and structure was evaluated. The best crRNA was selected, it couldtarget EGFR mRNA very specific and reduce its RNA level.

Keywords:

CRISPR-C13a , Brain stem glioma , EGFR , crRNA , CRISPR-RT