Development of a method for increasing gene-targeting efficiency in human cells

2014 Fiscal Year Final Research Report

• Project/Area Number: 25650006
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Molecular biology
• Research Institution: Kyoto University
• Principal Investigator: TAKEDA Shunichi 京都大学, 医学(系)研究科(研究院), 教授 (60188191)
• Co-Investigator(Renkei-kenkyūsha): SASANUMA Hiroyuki 京都大学, 大学院医学研究科, 准教授 (00531691)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Keywords: ゲノム編集 / CRISPR/Cas9 / DNA組換え / 標的組換え / DT40 / ニワトリBリンパ細胞 / プロテオーム解析

Outline of Final Research Achievements

The CRISPR/Cas9 system, an artificial restriction enzyme, dramatically increases the efficiency of genetic manipulation, such as knock-in of designed point mutations, in human cell lines. However, genetic manipulation in primary cells is still a formidable challenge, because the efficiency of homologous recombination (HR) between genomic DNA and transfected DNA is still very low despite the usage of the CRISPR/Cas9 system. This study is to further increase the efficiency of genetic manipulation. To this end, I have explored molecular mechanisms underlying the extremely efficient HR of a chicken B cell line, DT40. HR is a very complex biochemical reaction, which is carried out by more than 50 proteins including many unidentified proteins. Having a support of Grant-in-Aid for Challenging Exploratory Research, we have established methods of biochemically purifying proteins involved in HR. In future, I will purify proteins involved in HR from DT40 (wild-type and HR deficient cell lines).

Free Research Field

分子生物学