Analysis of Cell-cell interaction models with CRISPR library

2023 Fiscal Year Final Research Report

• Project/Area Number: 21K06945
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 49030:Experimental pathology-related
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: Kurata Morito 東京医科歯科大学, 大学院医歯学総合研究科, 講師 (40451926)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: 薬剤耐性 / CRISPR screening / 細胞間相互作用

Outline of Final Research Achievements

Indirect CRISPR screening for drug resistance with cell-cell interactions was invented. The photoconvertible fluorescent protein Dendra2 was inducted to supporting cells and explored the drug resistance responsible factors of supporting cells with CRISPR screenings. Random mutated supporting cells co-cultured with leukemic cells induced drug resistance with cell-cell interactions. Supporting cells responsible for drug resistance were isolated with green-to-red photoconversion, and 39 candidate genes were identified. Knocking out C9orf89, MAGI2, MLPH, or RHBDD2 in supporting cells reduced the ratio of apoptosis of cancer cells. Indirect CRISPR screening was established to isolate the responsible elements of cell-cell interactions. This screening method could reveal unknown mechanisms in all kinds of cell-cell interactions and it could be a platform for discovering new targets of drugs for conventional chemotherapies.

Free Research Field

がん生物学

Academic Significance and Societal Importance of the Research Achievements

従来は、薬剤曝露などの条件下でランダム変異を誘発するCRISPRライブラリーを用いて腫瘍細胞の選択的な生存/死滅を検出することにより、薬剤耐性候補遺伝子が同定されてきました。しかし、「がん微小環境」などの周辺環境からの細胞間相互作用による薬剤耐性の場合、細胞間相互作用による薬剤耐性の責任候補遺伝子の同定は困難でありました。これは、周囲の支持細胞にランダムな変異が誘導されても、支持細胞自身は薬剤下で選択的に生存/死滅することがないためです。そこで、我々は薬剤耐性を誘導できる支持細胞を分離するシステムを併用し、「間接的CRISPR screening」と名付けました。