Targeting Positive Regulators of Sonic Hedgehog Signaling Pathway in Medulloblastoma by Designing CRISPR/Cas9 Single Guide RNAs

 Medulloblastoma formation is importantly related to granular cell proliferation and differentiation, processes which are under the influence of sonic hedgehog (SHH) signaling. Exons of genes encoding different components of this signaling pathway (e.g., ligands, co-receptor, transcription factors, and target genes) were investigated to identify the proper single guide RNAs for selective targeting of positive regulators of the SHH pathway.

 The genomic DNA sequences of corresponding genes of several positive regulators of the SHH pathway (in Homo Sapiens), including SHH, SMO, GLI1, GLI2, MYCN, and MYC, were retrieved from the National Center for Biotechnology Information (NCBI) gene database. Next, the exon sequences of these genes were identified using protospacer adjacent motif (PAM) of NGG and Streptococcus pyogenes Cas9 nuclease and evaluated by CRISPOR software to select the best sgRNA for each target gene.

 The analyses revealed the best sgRNAs for the SHH, SMO, GLI1, GLI2, MYCN, and MYC genes targeted exon 1, -4, -4, -5, -2 and -2, respectively. Proper sgRNAs for targeting each exon in each gene were also identified.

 This study revealed possible specific exonic targets of components of the SHH signaling pathway through designing proper sgRNAs using the CRISPR/Cas9 genome editing approach.