Establishment of a precise deletion mutagenesis method using CRISPR/Cpf1 to enable gene therapy

2021 Fiscal Year Final Research Report

• Project/Area Number: 18K15054
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 48040:Medical biochemistry-related
• Research Institution: Tokyo Metropolitan Institute of Medical Science
• Principal Investigator: TAKAHASHI Gou 公益財団法人東京都医学総合研究所, 疾患制御研究分野, 研究員 (70802817)
• Project Period (FY): 2018-04-01 – 2022-03-31
• Keywords: CRISPR/Cpf1 / CRISPR-Cas12a / 欠失変異

Outline of Final Research Achievements

The objective of this study was to establish a precise deletion mutagenesis method by using CRISPR-Cas12a (Cpf1) to cleave two locations on a same chromosome. By amplicon sequencing to double cuts with Cas12a, I found that the percentage of precise deletions tended to be higher when the two guide RNAs were arranged to join inside a fragment to be deleted. Furthermore, in order to more easily and quantitatively detect the deletions, I established a digital PCR-based analysis system and succeeded in quantitatively detecting the deletions in hHPRT1 and dystrophin genes.

Free Research Field

遺伝子工学

Academic Significance and Societal Importance of the Research Achievements

本研究によって、ゲノム編集ツールの一つであるCas12aを利用して、ヒトゲノムに正確な欠失を導入することが可能であることが示唆された。高精度なゲノムへの欠失導入は、正確な遺伝子機能を解析できるほか、筋ジストロフィー症の遺伝子治療法であるエキソンスキッピングにも応用が期待される。一方で、遺伝子治療へCas12の欠失導入を応用するには、さらなる精度向上や実験動物を利用した検証が必要と考えられる。