Abstract
Neuroblastomas, the most coilllllon extracranial solid tumor found in children with a mean diagnosis age of 18 months, arise from malignant transformation of undifferentiated neuronal stem cells. These tumors often derive in the sympathetic nervous system. It has been suggested that tumors typically manifested in this area are more likely caused by spontaneous or predisposed genetic factors, rather than environmental exposure. However, most neuroblastoma cases are a result of spontaneous genetic changes such as chromosomal ploidy, activation of known oncogenes and tumorcell ploidy changes in somatic cells. Understanding the molecular bases that underlie these clinical outcomes may provide further insight into the disease pathogenesis. The abnormal expressiqn of a numoer of genes has already been linked to specific clinical diagnoses suggestfu.g that variance of specific genes may be causal in neuroblastoma mortality rates. One such gene, neuroblastorua-derived myelocytomatosis oncogene (MYCN), has been shown to positively correlat~ with poor neqroplastoma prognosis. MYCN is a transcription factor that forms heterodimers with transcriptiqnal co-factors, through helix loop helix lel.Jcine interactions, to target genes and affect downstream transcriptional activity. MYCN has been shown to regulate gene express~on as both an activator as well as a repressor in genes involved in fun<lamental cellular processes including cell cycling, differentiation, proliferati9n and apoptosis. To date an effective knockout model had not been established in human embryonic stem cell (hESC) lines, which will be necessary in order to transition from a mouse to a human model. The next generation Clustered Regularly Interspersed Palindromic Repeats (CRISPR) Cas9 nickase genome-editing tool can be used to create two single stranded breaks at specific locations in the genome. The cell repairs the introduced breaks by either non-homolo.gous end joining (NHEJ) or homology directed repair (HDR). NHEJ is an error prone method of recombination causing insertion or deletion (INDEL) mutations in the target gene leading to gene knockouts through frame shift mutations and premature stop codons. The use of the CRISPR-Cas9 nickase system increases the likelihood for NHEJ repair and consequently the opportunity for a gene disrupting INDEL, while decreasing the likelihood of off target effects. We hypothesize that through the use of CRISPR-Cas9 nickase MYCN knockout we can better understand how normal MYCN function in stem cells and later translate the results to understand abnormal MYCN gene expression effects neuroblastoma tumor formation and proliferation. The CRISPR-Cas9 nickase system was used to target the primary and secondary translational start sites of the MYCN gene in human embryonic kidney (HEK) 293T cells. To effectively target the specific location in the MYCN gene, and create INDEL mutations, two sgRNA were constructed complementary to the ATG region of the gene. Through sequencing ana g~nomic PCR analysis we show that deletions have been introduced to the MYCN gene at the translational start sites. These results support the targeted design of the CRISPR-Cas9 nickase system has accurately targeted and cleaved MYCN creating INDELs.