Title of Presentation

“Cutting Edge of Genome Editing Technology”

In recent years, much attention has been paid to genome editing, a technology that employs an artificial DNA cutting system to enable targeted genes to be modified at will. Genome editing makes use of the repair process of cut DNA in order to modify genes, enabling the introduction of mutations into genes (knock-out) and insertion of foreign genes (knock-in) even in micro-organisms, animals, and plants in which modification had previously been difficult. The first artificial DNA cutting enzyme to be developed was ZFN. This was followed in 2010 by the second-generation TALEN, which offered greater freedom in the selection of target sequences, with successful targeted genetic modifications reported in a range of organisms. In 2012, the third-generation CRISPR-Cas9 system was unveiled, astounding many researchers with its high levels of efficiency and simplicity. CRISPR-Cas9 is a method that utilizes the acquired immunity system of bacteria. A small chain of RNA (known as guide RNA) binds to the target sequence, and then forms a complex with the Cas9 nuclease, which cuts the DNA. The scientists who developed CRISPR-Cas9, Dr. Jennifer A. Doudna and Dr. Emmanuelle Charpentier, were honored with the Nobel Prize in Chemistry in 2020.

Our group began working on the production of ZFN more than ten years ago, and we have used ZFN to produce visualizations of genetic expression in animal embryos. We have also developed a highly active TALEN (Platinum TALEN) and reported gene disruptions and knock-ins in microorganisms, a variety of plants and animals, and cultured cells. Recently we have developed a new gene knock-in method (PITCh method) using the CRISPR-Cas9 and MMEJ repair route, and co-developed a modification technique (MhAX method) at the single base level in iPS cells. We have also succeeded in simultaneous gene knock-in into multiple gene loci, using the LoAD system that accumulates MMEJ route effectors.

This lecture introduces the basic principles of genome editing and directions in its technological development, and discusses the possibilities of genome editing for basic research and applied research (including development and quality enhancement of bio-fuels, drug discovery and gene therapy) in a variety of fields.

Profile

Web Site URL

http://www.mls.sci.hiroshima-u.ac.jp/smg/index.html

A brief Biography(As of April 1, 2020)

Mar 1989	B.Sc., School of Science, Hiroshima University
July 1992	Candidate, Graduate School of Science, Hiroshima University
Aug 1992	Assistant, Faculty of Science, Kumamoto University
June 2002	Lecturer, Graduate School of Science, Hiroshima University
Nov 2003	Associate Professor, Graduate School of Science, Hiroshima University
Apr 2004	Professor, Graduate School of Science, Hiroshima University
Apr 2019-Present	Professor, Graduate School of Integrated Sciences for Life, Hiroshima University
2017-Present	Professor, Joint Research Course for Next Generation Automotive Technology (concurrent appointment)
2019-Present	Director, Genome Editing Innovation Center, Hiroshima University (concurrent appointment)
2014-Present	Visiting Professor, Chromosome Engineering Research Center, Tottori University
2014-Present	Visiting Professor, Institute of Resource Development and Analysis, Kumamoto University
2016-Present	President, Japanese Society for Genome Editing

Details of selected Awards and Honors

Sep 2013	21st JB Award, The Japanese Society of Biochemistry
May 2017	Experimental Animals Outstanding Paper Prize
Dec 2018	Hiroshima Venture Incubation Awards (Individual) Gold Award

A list of selected Publications

Li J, Dong A, Saydaminova K, Chang HZ, Wang G, Ochiai H, Yamamoto T, Pertsinidis A. Single-molecule nanoscopy elucidates RNA Polymerase II transcription at single genes in live cells. Cell, 178(2): 491-506.e28, 2019

Nakade S, Mochida K, Kinii A, Nakamae K, Aida T, Tanaka K, Sakamoto N, Sakuma T, Yamamoto T. Biased genome editing using the local accumulation of DSB repair molecules system. Nature Communications, 9, 3270, 2018

Sakuma T, Nakade S, Sakane Y, Suzuki KT, Yamamoto T. MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nature Protocols, 11, 118–133, 2016

Ochiai H, Sugawara T, Yamamoto T. Simultaneous live imaging of the transcription and nuclear position of specific genes. Nucleic Acid Research, doi: 10.1093/nar/gkv624, 2015

Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y, Tanaka K, Sakuma T, Yamamoto T, Tanaka K. Cloning-free CRISPR/Cas system facilitates functional cassette knockin in mice. Genome Biology, 16: 87, 2015

Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M, Daimon T, Sezutsu H, Yamamoto T, Sakuma T, Suzuki K. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nature Communications, 5: 5560, 2014

Ochiai H, Miyamoto T, Kanai A, Hosoba K, Sakuma T, Kudo Y, Asami K, Ogawa A, Watanabe A, Kajii T, Yamamoto T, Matsuura S. TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome. Proc Natl Acad Sci U S A, 111: 1461-1466, 2014

Ochiai H, Sakamoto N, Fujita K, Nishikawa M, Suzuki KI, Matsuura S, Miyamoto T, Sakuma T, Shibata T, Yamamoto T. Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos. Proc Natl Acad Sci U S A, 109: 10915-10920, 2012

Yamamoto, T. Basic Principles and Applications of Genome Editing (Shokabo, 2018)

Yamamoto, T., ed. An Introduction to Genome Editing (Shokabo, 2016)