Seminar & Symposium/Admissions




講演者: 堀居 拓郎 (准教授、群馬大学 生体調節研究所附属 生体情報ゲノムリソースセンター)

演題: Genome and epigenome editing by CRISPR/Cas9 system


日時: 10月10日(水) 12:00-13:00

会場: 発生医学研究所1階 カンファレンス室



In this seminar, I will talk about two topics. First, I will present an efficient generation of conditional knockout mice using CRISPR/Cas9 system. Conditional knockout using Cre/loxP is essential for functional analysis of genes. CRISPR/Cas9 in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked loxP (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively. Sequential method improved the flox efficiency compared with ordinary simultaneous microinjection, leading to a high yield of offspring with floxed alleles.

Next, I will present an efficient method for targeted demethylation of specific sites using CRISPR/Cas9 and a repeating peptide array-based amplification (SunTag). We fused a catalytically inactive nuclease with an RNA-guided DNA-binding property (dCas9) to a repeating peptide array in order to recruit multiple copies of antibody-fused ten-eleven translocation (TET) 1 hydroxylase, which is involved in erasure of the methyl mark. This system strongly amplified the targeted demethylation capability compared to the system without peptide array-based amplification and can also be used for generation of model mice with epigenetic disorder.






担当分野: 細胞医学 中尾(内線:6804)

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