最先端研究セミナー

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

Abstract:

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.

References:

• Horii T, et al. Efficient generation of conditional knockout mice via sequential introduction of lox sites. Sci Rep. 7:7891 (2017).
• Morita S, et al. Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions. Nat Biotechnol. 34:1060-1065 (2016).
• Horii T, et al. Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering. Sci Rep. 4:4513 (2014).
• Horii T, et al. Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system. PeerJ. 1: e230 (2013).
• Horii T, et al. Generation of an ICF syndrome model by efficient genome editing of human induced pluripotent stem cells using the CRISPR system. Int J Mol Sci. 14:19774-81 (2013).

担当分野：　細胞医学　中尾（内線：6804）

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