| 研究実績の概要 |
In the fiscal year of 2014, we have generated a library of CRISPR/Cas9 sgRNAs to specifically target at the long non-coding UBE3A-ATS. A total of 32 different sequences of short guidance RNA (sgRNA) were designed and sub-cloned to the PX330 Cas9 plasmids. Next the sequences' activity were evaluated by Surveyor assay to identify the highest active ones, which presumably have the high affinity with the Cas9 protein. We then obtained the plasmid which encodes an inactive Cas9 without any cleavage function and we clone 12 sgRNAs based on the results of activity assay. Most of the sgRNA were targeted to the SNORD116 repeat sequence region and have multiple binding sites. The primary culture of neurons were prepared from ICR mouse and the plasmids were transfected into the primary cultured neurons. After three days of culture, we harvested the neuron and extract the RNA, then the expression level of UBE3A, UBE3A-ATS, were measured by real time qPCR. We found that 3 sgRNAs could modestly increase the expression of UBE3A and decrease the amount of UBE3A-ATS, suggesting the Crispr/Cas9 system might be a valid tool for future restore of paternal UBE3A expression.
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