CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations

2020 Fiscal Year Final Research Report

• Project/Area Number: 19K22380
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
• Research Institution: The University of Tokyo
• Principal Investigator: Tanaka Yosuke 東京大学, 医科学研究所, 助教 (10509087)
• Project Period (FY): 2019-06-28 – 2021-03-31
• Keywords: nCas9-CDA / TATAloxP

Outline of Final Research Achievements

In this study, we designed two cellular chain reaction systems that enable sequential sgRNA expression in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, the thymidine (T)-to-cytosine (C) substitutions in the scaffold region of sgRNA or TATA box containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, which leads to expression of next sgRNA. These reactions can proceed several times, thus generating cellular chain reactions. As a proof of the concept, we established a chain reaction through the repair of sgRNA scaffold mutations in 293T cells. Importantly, the results obtained in yeast or in vitro were not consistent with those in mammalian cells, suggesting that the in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.

Free Research Field

細胞生物学

Academic Significance and Societal Importance of the Research Achievements

今回の研究において、sgRNAの連鎖反応を哺乳類細胞で実現できた。本研究においては、この連鎖反応を細胞周期と連動した反応にできれば、細胞が何回分裂したかを計測可能になる。また、細胞内において制御できる連続反応を実現した本研究の技術は、様々な連続した現象・反応を細胞内で計測するような研究への応用が期待できる。本研究計画的には、マイルストーンの前半が終わったにすぎないが、本研究で実現できたsgRNAの連続反応は、前述したように様々な研究分野における新しい計測技術の基礎となるものであり、学術的意義は非常に大きい。