Single-cell level analysis of genome editing outcomes for medical applications

2021 Fiscal Year Final Research Report

• Project/Area Number: 20K21409
• 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: Tokyo Metropolitan Institute of Medical Science
• Principal Investigator: MIYAOKA Yuichiro 公益財団法人東京都医学総合研究所, 疾患制御研究分野, プロジェクトリーダー (20549521)
• Project Period (FY): 2020-07-30 – 2022-03-31
• Keywords: ゲノム編集 / HDR / NHEJ / Cas9 / HypaCas9 / １細胞 / SPiS

Outline of Final Research Achievements

Genome editing by CRISPR-Cas has already revolutionized science, medicine, agriculture, and other fields. However, genome editing outcomes have only been analyzed in cell populations but not in single cells. An understanding of editing outcomes in single cells would lead to precise estimation of the effects of genome editing, and enhance its applications.
For this purpose, we developed an efficient method to isolate genome edited cultured human cell clones using a specialized device for single cell dispensation. This allowed us to analyze more than 2,600 clones. We found strong binarity in the induction of genome editing (i.e., individual cells tend to undergo either full editing in all target alleles, or no editing at all even when the CRISPR components were expressed). Moreover, the induction of homologous recombination also tends to be binary to produce cells fully edited by recombination.

Free Research Field

分子生物学

Academic Significance and Societal Importance of the Research Achievements

あらゆる細胞の遺伝情報の改変を可能にするゲノム編集は、様々な分野に革命をもたらしている。しかし、これまでのゲノム編集結果解析手法は、細胞を集団として扱うことしかできず、細胞１つ１つに何が起きたかを知ることは難しかった。そこで我々は、細胞１つ１つを単離し、そこから増えてきた細胞を解析し、各細胞に起きたゲノム編集結果を明らかにした。その結果、全くゲノム編集が起こらない細胞が多く観察された。一方で、編集が起きた細胞では編集されうる遺伝子が全て編集されている場合が多かった。このように、ゲノム編集が全く起こらないか、起きるのであれば全ての標的遺伝子が編集される、という二者択一性を初めて明らかにした。