Development of genome editing technology with low off-target risk and high efficiency in human pluripotent stem cells

2017 Fiscal Year Final Research Report

• Project/Area Number: 15K06924
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: System genome science
• Research Institution: Keio University
• Principal Investigator: SONE Takefumi 慶應義塾大学, 医学部(信濃町), 訪問研究員 (00379091)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: ゲノム編集 / iPS細胞 / CRISPR / ノックイン / TALEN / 遺伝子治療 / 相同組換え / 疾患研究

Outline of Final Research Achievements

Genome editing technology has made it familiar for us to knock-out a gene, but it is still difficult to knock-in a certain DNA sequence precisely into a genomic locus. In this study, a simple and effective method was developed to construct a gene knock-in vector with a drug-resistant gene cassette. Using the vector in combination with TALEN of CRISPR expression vectors, responsible genes of iPSC lines derived from various disease patients were successfully corrected or those of control iPSC lines were modified to have mutation. Especially for CRISPR expression vector, an all-in-one construct was developed to express two sets of sgRNAs for two dual-nickase Cas9 pairs to cut two genomic loci at the same time for lower the risk of off-target effect. Combination of this vector with two knock-in vectors with two different drug-resistant gene cassette, a reliable homo knock-in system was also developed and confirmed for its feasibility.

Free Research Field

分子生物学