2020 Fiscal Year Annual Research Report
脳のコレステロール代謝を調節する長鎖ノンコーディングＲＮＡの機能解明
| Project/Area Number |
19F19386
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
SHIN JAE・WOO 国立研究開発法人理化学研究所, 生命医科学研究センター, チームリーダー (60553849)
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| Co-Investigator(Kenkyū-buntansha) |
PRABHU ANIKA 国立研究開発法人理化学研究所, 生命医科学研究センター, 外国人特別研究員
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| Project Period (FY) |
2019-11-08 – 2022-03-31
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| Keywords | CRISPR / iPSC differentiation / Non-coding RNA / Genomics / Neurodegeneration |
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| Outline of Annual Research Achievements |
The project planned in the beginning of the fellowship has beencontinued. The focus was to utilize CRISPR screening technologies toidentify the regulatory roles of non-coding RNAs in iPSC-derived neurons. Our access to patient-derived iPSCs from individuals suffering fromDravet Syndrome (DS), a disease caused by mutations in the sodium ionchannel subunit SCN1A, provided a unique and useful tool for thisproject. By establishing an interneuron differentiation protocol from theseiPS cells, we were able to perform single-cell sequencing to identifyrelevant and highly expressed non-coding RNAs. In addition, we usedpublicly available datasets, including HiC data, to identify cis-regulatoryelements in the nearby region of SCN1A. From this, we generated a list oftarget coding and non-coding genes for a custom CRISPRi screeninglibrary. We have also developed a DS iPSC reporter cell line, which will be used to measure SCNIA expression following knockdown of these target genes in the screen.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The project has focused on Dravet Syndrome,a neurodegenerativedisease caused by mutations in the gene SCN1A. We hypothesizedthat SCN1A is regulated by non -coding RNAs, and sought to screenfor non-coding RNAs that increase SCN1A expression and function.Summary of my progress to date:
◎I have completed Aim 1: I have identified the non-codingRNAs expressed in our iPSC-derived interneuron systemthrough single-cell sequencing and analysis.
◎I have partially completed Aim 2: I used the single-cellsequencing data to generate a custom sgRNA CRISPRi library.A lentivirus production method has been developed and willbe used to infect interneurons with these libraries. Additionally, a reporter cell line has been generated tomeasure changes in SCN1A expression. With these tools nowdeveloped and in hand, I will perform the primary screen tocomplete Aim 2 and identify any non-coding RNAs involved inthe regulation of the neurodegenerative disease Dravet Syndrome.
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| Strategy for Future Research Activity |
To finalize the study, the pooled CRISPRi screen will be performedand any positive hits (knockdown of genes that result in increasedSCN1A expression), will be further interrogated in a secondaryscreen to validate their role in SCN1A regulation.
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