Establishment of Purkinje cell specific CRISPR/Cas9 screening in whole cerebellum

2017 Fiscal Year Final Research Report

• Project/Area Number: 16K14553
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Neurophysiology / General neuroscience
• Research Institution: The University of Tokyo
• Principal Investigator: Watanabe Takaki 東京大学, 大学院医学系研究科(医学部), 特任助教 (90749798)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Keywords: 小脳 / プルキンエ細胞 / 登上線維 / CRISPR/Cas9 / シナプス / シナプス刈り込み

Outline of Final Research Achievements

I aimed to establish molecular screening for climbing fiber (CF)-Purkinje cell (PC) synapse elimination by knocking out a particular gene with CRISPR/Cas9 system in PCs of mouse whole cerebellum. As a result of testing several conditions, in utero electroporation with a double-electrode probe against both sides of cerebellum at embryonic day 11 showed the highest efficiency of gene transduction into PCs of whole cerebellum. I investigated the effects of knockout of a gene required for CF-PC synapse elimination with CRISPR/Cas9 by electrophysiologically counting the number of CFs on acute cerebellar slices. However, the knockout on PCs by either SpCas9 or eSpCas9 showed no significant impairment on CF-PC synapses. This result suggested a low efficiency of genome editing on PCs in this condition in spite of a high efficiency of gene transduction into whole cerebellar PCs.

Free Research Field

神経科学