| Budget Amount *help |
¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2017: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| 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.
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