The exploration of potential treatment for Angelman syndrome by CRISPR-Cas9 system

2014 Fiscal Year Research-status Report

• Project/Area Number: 26860955
• Research Institution: The Institute of Physical and Chemical Research
• Principal Investigator: LIU XIAOXI 独立行政法人理化学研究所, 脳科学総合研究センター, 研究員 (20709216)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: UBE3A / CRISPR

Outline of Annual Research Achievements

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.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

The experiment was going according to the initial proposal.

Strategy for Future Research Activity

We are going to test the combination of the sgRNA in its ability to recover the UBE3A.
Also the dosage will be also adjusted for optimal performance.