Seminar & Symposium/Entrance Exam info


Cutting edge Seminar



Speaker: Takuro Horii (Associate Professor, Biological Genome Resource Center, Gunma University Institute for Molecular and Cellular Regulation)


Title: Genome and epigenome editing by CRISPR/Cas9 system




Date&Time: 10 Oct. (Wed.) 2018, 12:00- 13:00

Venue: Conference Room(1F), IMEG



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.




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