#seiichi kanazawa

[jokesterino]

bandori oc, seiichi kanazawa !!!!

[k2i]

true/本当のこと (2007 - 2012) from 藤本隆行 on Vimeo.

A full-length uncut version of "true/本当のこと." A record of performance in Montreal 2011.

This performance premiered in 2007 and continued the world tour until 2012.

The main technical systems used in this performance; Myoelectric sensor >> UHF radio >> Max/MSP >> Sound sampling files >> Video >> Transducer (ButtKicker) + Scaffolding >> LED lighting + Circular truss >> Linear actuator + Electromagnet

Note: The first 6 min. 45 sec. Are almost silent. Then suddenly there is a loud noise ^_^

true/本当のこと Credit:

Direction/Lighting Design: Takayuki Fujimoto (Kinsei R&D/Dumb Type) 企画演出/照明: 藤本隆行

Choreography/Dance: Tsuyoshi Shirai (AbsT/baneto) 振付/出演: 白井 剛

Choreography/Dance/Text: Takao Kawaguchi (Dumb Type) 振付/出演/テキスト: 川口隆夫

Sound/Video/Visual Design: Takuya Minami (Softpad) 音/映像/ビジュアルデザイン: 南 琢也

Sound/Integrated Programming: Daito Manabe (Rhizomatiks) 音/統合制御プログラミング: 真鍋大度

Video/Programming/web: Satoshi Horii (Rhizomatiks) 映像/プログラミング/ウェブ: 堀井哲史

Table Design & Mechanics: Seiichi Saito・Motoi Ishibashi (Rhizomatiks) 装置: 齋藤精一・石橋 素

Myoelectric sensor & Oscillator support: Masaki Teruoka (VPP) 筋電センサー製作、振動子監修: 照岡正樹

Costume Design: Noriko Kitamura 衣装: 北村教子

Technical support: YCAM InterLab Color Kinetics Japan TamaTechLab Rhizomatiks DGN

Production management: HiWood - Koichiro Takagi, Ayako Tuchiya 制作: ハイウッド - 高樹光一郎、土屋彩子

Video editing: Takayuki Fujimoto

2007 01 Sep. World premiere Yamaguchi Center for Arts and Media [YCAM], StudioB Yamaguchi/Japan

08, 09 Dec. 21st Century Museum of Contemporary Art, Theater 21 Kanazawa/Japan

14-16 Dec. Yokohama Red Brick Warehouse No.1 Yokohama / Japan

2008 24-26 July Esplanade, Theatre Studio Singapore

13-15 Nov. Japan Society New York/USA

2009 06-09 Aug. Setagaya Public Theatre, Theatre Tram Tokyo/Japan

25, 26 Sep. Stadsschouwburg Amsterdam (SSBA) Amsterdam/Netherlands

29 Sep. Parktheater Eindhoven Eindhoven/Netherlands

03, 04 Oct. Tanzhaus NRW Dusseldorf Dusseldorf/Germany

09-10 Oct. Mousonturm Frankfurt/Germany

15-17 Oct. Maison de la culture du JAPON a Paris Paris / France

11, 12 Nov. Teatro Tom Jobim / Festival Panorama de Danca 2009 Rio de Janeiro/Brazil

18, 19 Nov. Teatro Peaulo Autran / SESC Pinheiros Sao Paulo/Brazil

2010 05 June Macao Cultural Centre, Small Auditorium Macao/China

18-20 June Hong Kong Cultural Centre, Studio Theatre Hong Kong/China (Organized by orleanlaiproject)

08, 09 Sep. EX-Alumix di Bolzano / TransART 2010 Bolzano/Italy

2011 27-29 Jan. Usine C Montreal/Canada

03-05 Feb. Cooperative Meduse/Mois-Multi Quebec/Canada

18-21 Mar. AI HALL/AI HALL Dance Collection vol.64 Hyogo/Japan

2012 08-09 Mar. LIG Art Hall Busan/Korea

Kinsei R&D: kinsei.asia/ facebook.com/KinseiRD/

[fictionalxlifexreality]

FictionalxLifexReality’s Wish List for the Kagome Xover Exchange @MizukixTsukiyomi

Here’s my wish list of animes/tv shows/movies/games and characters for the Kagome Xover Exchange! I’m open to any genres out there, although, I’m not too keen on anything that’s too dark or angsty.

I don’t mind receiving NFSW material, but if you ask me to do one, I can’t really say I can do it. I’ll do my best, but I don’t really know how to write one, let alone draw one, without messing up too horribly. 

These are all the animes/mangas I’ve watched and read, movies and TV shows I’ve enjoyed watching and games my brothers and I, well mostly my brothers, have played over the years, that I think would be interesting for a xover with Kagome.

Animes/Mangas:

07-Ghost: Teito Klein, Frau (Zehel)

Acchi Kocchi: Otonashi Io

Akagami no Shirayukihime: Zen Wistaria, Izana Wistaria, Obi

Amatsuki: Rikugō Tokidoki, Shinonome Kon, Kuchiha

Aoharu x Kikanjuu: Yukimura Tōru, Matsuoka Masamune

Black Blood Brothers: Mochizuki Jiro

Black Cat: Train Heartnet

Blood+: Haji, Solomon Goldsmith

Barakamon: Handa Seishū, Kotoishi Naru

Brave 10: Kirigakure Saizō, Sarutobi Sasuke

Bungou Stray Dogs: Dazai Osamu, Kunikida Doppo, Nakajima Atsushi

Cardcaptor Sakura: Kinomoto Tōya, Tsukishiro Yukito (Yue)

Clamp School Detectives: Imonoyama Nokoru, Ijyuin Akira, Takamura Suoh

Code:Breaker: Ōgami Rei

Cooking Master Boy/Chuuka Ichiban!: Liu Mao Xing, Lan Fei Hong

D.N.Angel: Niwa Daisuke, Dark Mousy, Hiwatari Satoshi, Krad Hikari

D.Gray-Man: Allen Walker, Kanda Yū, Lavi, Lenalee Lee

Dantalian no Shoka: Huey Anthony Disward, Dalian

Death Note: L, Yagami Raito

Durarara!!!: Heiwajima Shizuo, Orihara Izaya

Eyeshield 21: Hiruma Yōichi, Kobayakawa Sena, Shin Seijūrō

Fairy Tail: Natsu Dragneel, Gray Fullbuster

Free!: Nanase Haruka, Tachibana Rin, Matsuoka Rin

Fruits Basket: Sohma Yuki, Sohma Hatsuharu

Fushigi Yuugi: Hotohori, Tamahome, Nuriko, Mitsukake

Gakuen Alice: Hyūga Natsume

Gakuen Heaven: Itō Keita, Endō Kazuki, Niwa Tetsuya, Nakajima Hideaki, Shinomiya Kōji

Gakuen Mokushiroku/Highschool of the Dead: Komuro Takashi

GetBackers: Mido Ban, Amano Ginij, Fuyuki Shido, Fūchōin Kazuki, Kakei Jūbei, Akabane Kuro'udō

Ghost Hunt: Shibuya Kazuya (Naru, Oliver Davis)

Gosick: Kūjo Kazuya, Victorique de Blois

Gravitation: Sakuma Ryuichi, Shindou Shuichi, Eiri Yuki

Hagane no Renkinjutsushi/Fullmetal Alchemist: Edward Elric, Alphonse Elric, Roy Mustang

Hakkenden: Touhou Hakken Ibun: Inuzuka Shino, Inukawa Sōsuke, Satomi Riō, Inukai Genpachi, Inusaka Keno, Inumura Daikaku

Hakuouki: Hijikata Toshizō, Okita Souji, Hajime Saitou

Hamatora: Nice, Murasaki, Hajime, Art

Hatenkō Yugi: Alzeid, Rahzel Anadis (Rahzenshia Rose), Baroqueheat Anadis

Hayate no Gotoku: Ayasaki Hayate

Hetalia: North Italy (Italia Veneziano, Feliciano Vargas), Germany (Ludwig), Japan (Honda Kiku), China (Wáng Yào), America (Alfred F. Jones), England (Arthur Kirkland), France (Francis Bonnefoy), Russia (Ivan Braginsky), South Italy (Romano, Lovino Vargas), Spain (Antonio Fernández Carriedo)

Hikaru no Go: Shindō Hikaru, Fujiwarano Sai, Tōya Akira

Hyouka: Oreki Hōutarō

Junjō Romantica: Takahashi Misaki, Akihiko Usami

Kamisama Hajimemashita: Tomoe, Mizuki

Karneval: Gareki, Nai Muhinyi, Yogi, Tsukumo, Hirato, Akari, Karoku Arumerita

Kateikyoushi Hitman Reborn!: Sawada Tsunayoshi, Reborn, Hibari Kyoya, Rokudō Mukuro

Kini’iro no Corda/La Corda D’Oro: 

Primo Passo: Lili, Tsukimori Len, Yunoki Azuma, Hihara Kazuki, Shimizu Keiichi, Ousaki Shinobu, Kanazawa Hiroto

Secondo Passo: Kaji Aoi, Etō Kiriya, Kira Akihiko

Blue♪Sky: Kisaragi Ritsu, Kisaragi Kyoya

Kyoukai no Kanata: Kanbara Akihito, Nase Hiroomi

Kyou Kara Maoh!: Shibuya Yuuri, Wolfram von Bielefelt, Gwendal von Voltaire, Conrart Weller

La storia della Arcana Famiglia/Arcana Famiglia: Libertà, Nova, Luca, Jolly, Pace, Ash

Love Stage!!: Sena Izumi, Ichijou Ryōma, Sena Shougo, Sagara Rei

Loveless: Agatsuma Soubi, Aoyagi Ritsuka, Aoyagi Seimei

Makai Ouji: Devils and Realist: William Twining, Dantalion, Sytry, Camio

Mamotte! Lollipop: Zero, Ichî

Matantei Loki Ragnarok: Loki, Heimdall (Higashiyama Kazumi)

Mobile Suit Gundam Wing: Heero Yuy, Duo Maxwell

Mushishi: Ginko

Nabari no Ou: Rokujō Miharu, Yoite

Natsume Yuujinchou: Natsume Takashi, Nyanko-sensei (Madara), Tanuma Kaname, Natori Shuuichi, Matoba Seiji

Nightwalker: The Midnight Detective: Tatsuhiko Shido

No. 6: Shion, Nezumi

Noblesse: The Awakaning: Rai (Cadis Etrama Di Raizel), Frankenstein

Noragami: Yato, Yukine

Nurarihyon no Mago: Nura Rikuo

Ōkiku Furikabutte/Big Windup!: Mihashi Ren, Abe Takaya

Pandora Hearts: Oz Vessalius, Gilbert Nightray, Xerxes Break

Pet Shop of Horrors: Count D, Leon Orcot

Prince of Stride: Alternative: Yagami Riku, Fujiwara Takeru, Kuga Kyōsuke, Hasekura Heath

Ranma 1/2: Saotome Ranma

Rave Master: Haru Glory, Hamrio Musica

Recca no Honoo: Hanabishi Recca, Mikagami Tokiya

Rokka no Yuusha: Adlet Maia, Goldof Auora

Rurouni Kenshin: Himura Kenshin, Shinomori Aoshi

Saiunkoku Monogatari: Shi Seiran, Shi Ryuuki

Saiyuki:  Genjō Sanzō, Son Gokū, Cho Hakkai, Sha Gojō

Shigatsu wa Kimi no Uso: Arima Kousei, Miyazono Kaori

Shinrei Tantei Yakumo: Saitou Yakumo

Shokugeki no Soma: Yukihira Soma, Hayama Akira, Takumi Aldini, Kurokiba Ryō

Shounen Onmyouji: Abe no Masahiro, Guren/Touda

Shugo Chara!: Tsukiyomi Ikuto, Fujisaki Nagihiko

Skip Beat!: Tsuruga Ren

Soredemo Sekai wa Utsukushii: Livius Orvinus Ifrikia

S · A: Special A: Takishima Kei

Sakamoto desu ga?/Haven't You Heard? I'm Sakamoto: Sakamoto, Hayabusa Shou

Sword Art Online: Kirito (Kirigaya Kazuto)

Tactics: Kantarō, Haruka

Tanaka-kun wa Itsumo Kedaruge: Tanaka, Oota

Tegami Bachi/Letter Bee: Lag Seeing, Gauche Suede, Sylvette Suede

Tennis no Ouji-sama/Prince of Tennis: Echizen Ryōma, Tezuka Kunimitsu, Fuji Shūsuke, Yukimura Seiichi, Sanada Genichirō, Shiraishi Kuranosuke, Echizen Ryoga

Touken Ranbu: Izuminokami Kanesada, Kogitsunemaru, Nakigitsune, Munechika Mikazuki

Tsubasa Reservoir Chronicle: Fai D. Flowright, Kurogane

Tsuritama: Sanada Yuki, Usami Natsuki, Haru, Akira Agarkar Yamada

Uta no☆Prince-sama♪:

Maji Love 1000%: Ittoki Otoya, Ichinose Tokiya, Jingujin Ren, Hijirikawa Masato

Maji Love 2000%: Kurosaki Ranmaru, Camus

Vampire Knight: Kiryū Zero, Kuran Kaname

Wolf's Rain: Kiba

X/1999: Shirō Kamui

xxxHolic: Watanuki Kimihiro, Dōmeki Shizuka, Ichihara Yuuko

Yami no Matsuei: Tsuzuki Asato, Kurosaki Hisoka, Muraki Kazutaka

Yu Yu Hakusho: Minamino Shūichi (Yōko Kurama), Hiei

Yu-Gi-Oh: Mutou Yugi, Yami Yugi (Atem), Kaiba Seto

Yumeiro Patissiere: Kashino Makoto, Andou Sennousuke, Hanabusa Satsuki

Yuri!!! On Ice: Katsuki Yuri, Viktor Nikiforov, Yuri Plisetsky

Zombie-Loan: Akatsuki Chika, Tachibana Shito, Kita Michiru

TV Shows:

Pokémon series (pick your favorite series!)

Avatar: Last Airbender 

BBC Sherlock: Sherlock Holmes, John Watson

BBC Merlin: Merlin, Arthur Pendragon, Guinevere

Supernatural: Dean Winchester, Sam Winchester

The Walking Dead

Movies:

Hayao Miyazaki’s Films

Laputa: Castle in the Sky: Pazu, Sheeta (Princess Lusheeta Toel Ur Laputa)

Princess Mononoke: Ashitaka

Sen to Chihiro no Kamikakushi/Spirited Away: Haku (Nigihayami Kohaku Nushi)

Howl’s Moving Castle: Howl Jenkins Pendragon

Tales of Earthsea: Arren, Teru

Ponyo on the Cliff by the Sea: Sōsuke, Ponyo, Lisa

The Secret World of Arrietty: Arrietty Clock, Shō

Battle Royale: Nanahara Shuya, Kiriyama Kazuo

Percy Jackson and the Olympians: Percy Jackson

Miss Peregrine’s Home for Peculiar Children

The Chronicles of Narnia: Peter Pevensie

Harry Potter

The Lord of the Rings: Legolas, Aragorn

The Hobbit: Bilbo Baggins

The Legend of Tarzan (2016)

The Jungle Book (2016): Mowgli, Baloo, Bagheera

Cinderella (2015)

Beauty and the Beast (2017)

Rise of the Guardians: Jack Frost

Frozen: Elsa

The Little Mermaid: Ariel

Mulan

How to Train Your Dragon: Hiccup Horrendous Haddock III, Toothless

Games:

Tales of the Abyss: Jade Curtiss

Joker/Clover/Heart no Kuni no Alice: Peter White, Ace, Blood Dupre, Tweedle Dee and Dum, Julius Monrey, Boris, Airay, Joker, Gray Ringmarc, Nightmare Gottschalk

Devil May Cry: Dante, Vergil

Persona series

The Last Story: Zael (Elza), Yurick (Yuris), Therius (Tasha)

Kingdom Hearts: Sora, Riku, Roxas

Legend of Zelda: Link

Final Fantasy: Chocobos! Moogles!

VII: Zack Fair, Sepiroth, Aerith Gainsborough, Cloud Strife, Vincent Valentine

VIII: Squall Leonheart

X: Tidus, Yuna

XIII: Lightning, Noel, Caius, Yeul

XV: Noctis Lucis Caelum, Prompto, Ignis, Gladiolus

Folklore: Ellen, Keats

Assassin’s Creed

Resident Evil (the movie franchise or the animated films or the game, your pick!): Alice (movie exclusive character), Leon S. Kennedy, Claire Redfield, Chris Redfield 

Silent Hill (the movies or the games, your pick!): 

1: Alessa Gillespie (also in the Silent Hill films)

2: James Sunderland

3: Heather Mason (aka Sharon Da Silva in the Silent Hill films)

[abbkine]

BioAdvisers said on Biotech Advisers

Visualization of CRISPR-Cas9 genetic-engineering technique

Researchers at Kanazawa University and the University of Tokyo report in Nature Communications the visualization of the dynamics of ‘molecular scissors’ — the main mechanism of the CRISPR-Cas9 genetic-engineering technique. This study provides unprecedented details about the functional dynamics of CRISPR-Cas9, and highlights the potential of HS-AFM to elucidate the action mechanisms of RNA-guided effector nucleases from distinct CRISPR-Cas systems.

CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.Selection of the site to be cut is done by a ‘guide RNA’ molecule bound to the Cas9 protein.  Now, a team of researchers led by Mikihiro Shibata from Kanazawa University and Osamu Nureki from the University of Tokyo has visualized the dynamics of the CRISPR-Cas9 complex, in particular how it cuts DNA, providing valuable insights into the CRISPR-Cas9-mediated DNA cleavage mechanism.

From left to right: Cas9 alone (apo-Cas9), Cas9 bound to RNA (Cas9–RNA), Cas9–RNA bound to its single-stranded DNA target (Cas9–RNA–DNA), Cas9–RNA bound to a partial DNA duplex (Cas9–RNA–DNA) and Cas9–RNA bound to its double-stranded DNA target (Cas9–RNA–DNA).

For their visualization studies, the scientists used high-speed atomic-force microscopy (HS-AFM), a method for imaging surfaces.  A surface is probed by moving a tiny cantilever over it; the force experienced by the probe can be converted into a height measure.  A scan of the whole surface then results in a height map of the sample.  The high-speed experimental set-up of Shibata and colleagues enabled extremely fast, repeated scans — convertible into movies — of the biomolecules taking part in the molecular scissoring action.

Fluctuations of the nuclease domain are indicated by magenta arrows. The cleavage products released from Cas9–RNA are indicated by blue arrows.

First, the scientists compared Cas9 without and with RNA attached (Cas9–RNA).  They found that the former was able to flexibly adopt various conformations, while the latter has a fixed, two-lobe structure, highlighting the conformational-stabilization ability of the guide RNA.  Then, Shibata and colleagues looked at how the stabilized Cas9–RNA complex targets DNA.  They confirmed that it binds to a pre-selected protospacer adjacent motif (PAM) site in the DNA.  A PAM is a short nucleotide sequence located next to the DNA’s target site, which is complementary to the guide RNA.

The research team’s high-speed movies further revealed that targeting (‘DNA interrogation’) is achieved through 3D diffusion of the Cas9–RNA complex.  Finally, the researchers managed to visualize the dynamics of the cleavage process itself: they observed how the region of ‘molecular scissors’ undergoes conformational fluctuations after Cas9–RNA locally unwinds the double-stranded DNA.

Article Title: Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy Journal: Nature Communications Authors: Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi, and Osamu Nureki

Doi: 10.1038/s41467-017-01466-8

Funders The Kao Foundation for Arts and Science, The Brain Science Foundation, JST/PRESTO, JST/CREST, The Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED, JSPS KAKENHI

[abbkineeu]

New Post has been published on Biotech Advisers

New Post has been published on http://www.bioadvisers.com/visualization-crispr-cas9-genetic-engineering-technique/

Visualization of CRISPR-Cas9 genetic-engineering technique

Researchers at Kanazawa University and the University of Tokyo report in Nature Communications the visualization of the dynamics of ‘molecular scissors’ — the main mechanism of the CRISPR-Cas9 genetic-engineering technique. This study provides unprecedented details about the functional dynamics of CRISPR-Cas9, and highlights the potential of HS-AFM to elucidate the action mechanisms of RNA-guided effector nucleases from distinct CRISPR-Cas systems.

CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.Selection of the site to be cut is done by a ‘guide RNA’ molecule bound to the Cas9 protein.  Now, a team of researchers led by Mikihiro Shibata from Kanazawa University and Osamu Nureki from the University of Tokyo has visualized the dynamics of the CRISPR-Cas9 complex, in particular how it cuts DNA, providing valuable insights into the CRISPR-Cas9-mediated DNA cleavage mechanism.

From left to right: Cas9 alone (apo-Cas9), Cas9 bound to RNA (Cas9–RNA), Cas9–RNA bound to its single-stranded DNA target (Cas9–RNA–DNA), Cas9–RNA bound to a partial DNA duplex (Cas9–RNA–DNA) and Cas9–RNA bound to its double-stranded DNA target (Cas9–RNA–DNA).

For their visualization studies, the scientists used high-speed atomic-force microscopy (HS-AFM), a method for imaging surfaces.  A surface is probed by moving a tiny cantilever over it; the force experienced by the probe can be converted into a height measure.  A scan of the whole surface then results in a height map of the sample.  The high-speed experimental set-up of Shibata and colleagues enabled extremely fast, repeated scans — convertible into movies — of the biomolecules taking part in the molecular scissoring action.

Fluctuations of the nuclease domain are indicated by magenta arrows. The cleavage products released from Cas9–RNA are indicated by blue arrows.

First, the scientists compared Cas9 without and with RNA attached (Cas9–RNA).  They found that the former was able to flexibly adopt various conformations, while the latter has a fixed, two-lobe structure, highlighting the conformational-stabilization ability of the guide RNA.  Then, Shibata and colleagues looked at how the stabilized Cas9–RNA complex targets DNA.  They confirmed that it binds to a pre-selected protospacer adjacent motif (PAM) site in the DNA.  A PAM is a short nucleotide sequence located next to the DNA’s target site, which is complementary to the guide RNA.

The research team’s high-speed movies further revealed that targeting (‘DNA interrogation’) is achieved through 3D diffusion of the Cas9–RNA complex.  Finally, the researchers managed to visualize the dynamics of the cleavage process itself: they observed how the region of ‘molecular scissors’ undergoes conformational fluctuations after Cas9–RNA locally unwinds the double-stranded DNA.

Article Title: Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy Journal: Nature Communications Authors: Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi, and Osamu Nureki

Doi: 10.1038/s41467-017-01466-8

Funders The Kao Foundation for Arts and Science, The Brain Science Foundation, JST/PRESTO, JST/CREST, The Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED, JSPS KAKENHI

[bioadvisers]

Bioadvisers shared on Biotech Advisers

Bioadvisers shared on http://www.bioadvisers.com/visualization-crispr-cas9-genetic-engineering-technique/

Visualization of CRISPR-Cas9 genetic-engineering technique

Researchers at Kanazawa University and the University of Tokyo report in Nature Communications the visualization of the dynamics of ‘molecular scissors’ — the main mechanism of the CRISPR-Cas9 genetic-engineering technique. This study provides unprecedented details about the functional dynamics of CRISPR-Cas9, and highlights the potential of HS-AFM to elucidate the action mechanisms of RNA-guided effector nucleases from distinct CRISPR-Cas systems.

CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.Selection of the site to be cut is done by a ‘guide RNA’ molecule bound to the Cas9 protein.  Now, a team of researchers led by Mikihiro Shibata from Kanazawa University and Osamu Nureki from the University of Tokyo has visualized the dynamics of the CRISPR-Cas9 complex, in particular how it cuts DNA, providing valuable insights into the CRISPR-Cas9-mediated DNA cleavage mechanism.

From left to right: Cas9 alone (apo-Cas9), Cas9 bound to RNA (Cas9–RNA), Cas9–RNA bound to its single-stranded DNA target (Cas9–RNA–DNA), Cas9–RNA bound to a partial DNA duplex (Cas9–RNA–DNA) and Cas9–RNA bound to its double-stranded DNA target (Cas9–RNA–DNA).

For their visualization studies, the scientists used high-speed atomic-force microscopy (HS-AFM), a method for imaging surfaces.  A surface is probed by moving a tiny cantilever over it; the force experienced by the probe can be converted into a height measure.  A scan of the whole surface then results in a height map of the sample.  The high-speed experimental set-up of Shibata and colleagues enabled extremely fast, repeated scans — convertible into movies — of the biomolecules taking part in the molecular scissoring action.

Fluctuations of the nuclease domain are indicated by magenta arrows. The cleavage products released from Cas9–RNA are indicated by blue arrows.

First, the scientists compared Cas9 without and with RNA attached (Cas9–RNA).  They found that the former was able to flexibly adopt various conformations, while the latter has a fixed, two-lobe structure, highlighting the conformational-stabilization ability of the guide RNA.  Then, Shibata and colleagues looked at how the stabilized Cas9–RNA complex targets DNA.  They confirmed that it binds to a pre-selected protospacer adjacent motif (PAM) site in the DNA.  A PAM is a short nucleotide sequence located next to the DNA’s target site, which is complementary to the guide RNA.

The research team’s high-speed movies further revealed that targeting (‘DNA interrogation’) is achieved through 3D diffusion of the Cas9–RNA complex.  Finally, the researchers managed to visualize the dynamics of the cleavage process itself: they observed how the region of ‘molecular scissors’ undergoes conformational fluctuations after Cas9–RNA locally unwinds the double-stranded DNA.

Article Title: Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy Journal: Nature Communications Authors: Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi, and Osamu Nureki

Doi: 10.1038/s41467-017-01466-8

Funders The Kao Foundation for Arts and Science, The Brain Science Foundation, JST/PRESTO, JST/CREST, The Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED, JSPS KAKENHI

[scitechnetwork]

Kanazawa University research: Genetic engineering mechanism visualized

Source: Kanazawa University

(Kanazawa, 10 November 2017) Researchers at Kanazawa University and the University of Tokyo report in Nature Communications the visualization of the dynamics of ‘molecular scissors’ — the main mechanism of the CRISPR-Cas9 genetic-engineering technique.

One of the techniques used in genetic engineering — the process of artificially modifying the genome of a living organism — involves the so-called CRISPR-Cas9 nuclease system.  Using this system, a cell’s DNA can be cut at a desired site, where genes can be deleted or added.  Selection of the site to be cut is done by a ‘guide RNA’ molecule bound to the Cas9 protein. Now, a team of researchers led by Mikihiro Shibata from Kanazawa University and Osamu Nureki from the University of Tokyo has visualized the dynamics of the CRISPR-Cas9 complex, in particular how it cuts DNA, providing valuable insights into the CRISPR-Cas9-mediated DNA cleavage mechanism.

For their visualization studies, the scientists used high-speed atomic-force microscopy (HS-AFM), a method for imaging surfaces.  A surface is probed by moving a tiny cantilever over it; the force experienced by the probe can be converted into a height measure. A scan of the whole surface then results in a height map of the sample.  The high-speed experimental set-up of Shibata and colleagues enabled extremely fast, repeated scans — convertible into movies — of the biomolecules taking part in the molecular scissoring action.

First, the scientists compared Cas9 without and with RNA attached (Cas9–RNA).  They found that the former was able to flexibly adopt various conformations, while the latter has a fixed, two-lobe structure, highlighting the conformational-stabilization ability of the guide RNA.  Then, Shibata and colleagues looked at how the stabilized Cas9–RNA complex targets DNA. They confirmed that it binds to a pre-selected protospacer adjacent motif (PAM) site in the DNA.  A PAM is a short nucleotide sequence located next to the DNA’s target site, which is complementary to the guide RNA.

The research team’s high-speed movies further revealed that targeting (‘DNA interrogation’) is achieved through 3D diffusion of the Cas9–RNA complex.  Finally, the researchers managed to visualize the dynamics of the cleavage process itself: they observed how the region of ‘molecular scissors’ undergoes conformational fluctuations after Cas9–RNA locally unwinds the double-stranded DNA .

The work of Shibata advances our understanding of the CRISPR-Cas9 genome-editing mechanism.  In the words of the researchers: “… this study provides unprecedented details about the functional dynamics of CRISPR-Cas9, and highlights the potential of HS-AFM to elucidate the action mechanisms of RNA-guided effector nucleases from distinct CRISPR-Cas systems.”

Background

CRISPR-Cas9

CRISPR, short for “clustered regularly interspaced short palindromic repeats”, refers to a set of bacterial DNA sequences containing fragments of the DNA of viruses having earlier attacked the bacteria.  These fragments are used by the bacteria to prevent further attacks by the same viruses.  “Cas” refers to CRISPR-associated genes; “Cas9” is a CRISPR-associated protein with two nuclease domains (A nuclease is an enzyme capable of cleaving nucleic acids, organic molecules present in DNA and RNA).

In recent years, a genetic-engineering technique where a CRISPR-Cas9 complex acts as ‘molecular scissors’ has been developed; the Cas9 nuclease binds to a guide RNA molecule that contains information about the DNA site to target.  Using high-speed atomic force microscopy, Mikihiro Shibata from Kanazawa University and colleagues have now studied the dynamics of the CRISPR-Cas9 complex in great detail. 

Atomic force microscopy

Atomic force microscopy (AFM) is an imaging technique in which the image is formed by scanning a surface with a very small tip. Horizontal scanning motion of the tip is controlled via piezoelectric elements, while vertical motion is converted into a height profile, resulting in a height distribution of the sample’s surface.  As the technique does not involve lenses, its resolution is not restricted by the so-called diffraction limit.  In a high-speed setup, AFM can be used to produce movies of a sample’s evolution in real time.  High-speed AFM has been used successfully to study protein dynamics, for example myosin V walking on an actin filament, the photo-induced conformational change of bacteriorhodopsin, and the degradation of cellulose.  Shibata and colleagues have now applied the high-speed AFM technique for visualizing the dynamics of DNA cleavage by CRISPR-Cas9.

Reference

Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi & Osamu Nureki. Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy. Nature Communications, 10th November 2017.

DOI: 10.1038/s41467-017-01466-8

https://www.nature.com/articles/ s41467-017-01466-8

Figure 1. [Fig. 1B of the paper]

Structures of Cas9.  From left to right: Cas9 alone (apo-Cas9), Cas9 bound to RNA (Cas9–RNA), Cas9–RNA bound to its single-stranded DNA target (Cas9–RNA–DNA), Cas9–RNA bound to a partial DNA duplex (Cas9–RNA–DNA) and Cas9–RNA bound to its double-stranded DNA target (Cas9–RNA–DNA).

Movie 1. [Supplementary Movie 5 of the paper]

HS-AFM movies of DNA cleavage by Cas9–RNA. Fluctuations of the nuclease domain are indicated by magenta arrows. The cleavage products released from Cas9–RNA are indicated by blue arrows.

Provided by: Kanazawa University

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New Post has been published on Biotech Advisers

New Post has been published on http://www.bioadvisers.com/visualization-crispr-cas9-genetic-engineering-technique/

Visualization of CRISPR-Cas9 genetic-engineering technique

Researchers at Kanazawa University and the University of Tokyo report in Nature Communications the visualization of the dynamics of ‘molecular scissors’ — the main mechanism of the CRISPR-Cas9 genetic-engineering technique. This study provides unprecedented details about the functional dynamics of CRISPR-Cas9, and highlights the potential of HS-AFM to elucidate the action mechanisms of RNA-guided effector nucleases from distinct CRISPR-Cas systems.

CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.Selection of the site to be cut is done by a ‘guide RNA’ molecule bound to the Cas9 protein.  Now, a team of researchers led by Mikihiro Shibata from Kanazawa University and Osamu Nureki from the University of Tokyo has visualized the dynamics of the CRISPR-Cas9 complex, in particular how it cuts DNA, providing valuable insights into the CRISPR-Cas9-mediated DNA cleavage mechanism.

From left to right: Cas9 alone (apo-Cas9), Cas9 bound to RNA (Cas9–RNA), Cas9–RNA bound to its single-stranded DNA target (Cas9–RNA–DNA), Cas9–RNA bound to a partial DNA duplex (Cas9–RNA–DNA) and Cas9–RNA bound to its double-stranded DNA target (Cas9–RNA–DNA).

For their visualization studies, the scientists used high-speed atomic-force microscopy (HS-AFM), a method for imaging surfaces.  A surface is probed by moving a tiny cantilever over it; the force experienced by the probe can be converted into a height measure.  A scan of the whole surface then results in a height map of the sample.  The high-speed experimental set-up of Shibata and colleagues enabled extremely fast, repeated scans — convertible into movies — of the biomolecules taking part in the molecular scissoring action.

Fluctuations of the nuclease domain are indicated by magenta arrows. The cleavage products released from Cas9–RNA are indicated by blue arrows.

First, the scientists compared Cas9 without and with RNA attached (Cas9–RNA).  They found that the former was able to flexibly adopt various conformations, while the latter has a fixed, two-lobe structure, highlighting the conformational-stabilization ability of the guide RNA.  Then, Shibata and colleagues looked at how the stabilized Cas9–RNA complex targets DNA.  They confirmed that it binds to a pre-selected protospacer adjacent motif (PAM) site in the DNA.  A PAM is a short nucleotide sequence located next to the DNA’s target site, which is complementary to the guide RNA.

The research team’s high-speed movies further revealed that targeting (‘DNA interrogation’) is achieved through 3D diffusion of the Cas9–RNA complex.  Finally, the researchers managed to visualize the dynamics of the cleavage process itself: they observed how the region of ‘molecular scissors’ undergoes conformational fluctuations after Cas9–RNA locally unwinds the double-stranded DNA.

Article Title: Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy Journal: Nature Communications Authors: Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi, and Osamu Nureki

Doi: 10.1038/s41467-017-01466-8

Funders The Kao Foundation for Arts and Science, The Brain Science Foundation, JST/PRESTO, JST/CREST, The Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED, JSPS KAKENHI

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Bioadvisers shared on Biotech Advisers

Bioadvisers shared on http://www.bioadvisers.com/visualization-crispr-cas9-genetic-engineering-technique/

Visualization of CRISPR-Cas9 genetic-engineering technique

Researchers at Kanazawa University and the University of Tokyo report in Nature Communications the visualization of the dynamics of ‘molecular scissors’ — the main mechanism of the CRISPR-Cas9 genetic-engineering technique. This study provides unprecedented details about the functional dynamics of CRISPR-Cas9, and highlights the potential of HS-AFM to elucidate the action mechanisms of RNA-guided effector nucleases from distinct CRISPR-Cas systems.

CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.Selection of the site to be cut is done by a ‘guide RNA’ molecule bound to the Cas9 protein.  Now, a team of researchers led by Mikihiro Shibata from Kanazawa University and Osamu Nureki from the University of Tokyo has visualized the dynamics of the CRISPR-Cas9 complex, in particular how it cuts DNA, providing valuable insights into the CRISPR-Cas9-mediated DNA cleavage mechanism.

From left to right: Cas9 alone (apo-Cas9), Cas9 bound to RNA (Cas9–RNA), Cas9–RNA bound to its single-stranded DNA target (Cas9–RNA–DNA), Cas9–RNA bound to a partial DNA duplex (Cas9–RNA–DNA) and Cas9–RNA bound to its double-stranded DNA target (Cas9–RNA–DNA).

For their visualization studies, the scientists used high-speed atomic-force microscopy (HS-AFM), a method for imaging surfaces.  A surface is probed by moving a tiny cantilever over it; the force experienced by the probe can be converted into a height measure.  A scan of the whole surface then results in a height map of the sample.  The high-speed experimental set-up of Shibata and colleagues enabled extremely fast, repeated scans — convertible into movies — of the biomolecules taking part in the molecular scissoring action.

Fluctuations of the nuclease domain are indicated by magenta arrows. The cleavage products released from Cas9–RNA are indicated by blue arrows.

First, the scientists compared Cas9 without and with RNA attached (Cas9–RNA).  They found that the former was able to flexibly adopt various conformations, while the latter has a fixed, two-lobe structure, highlighting the conformational-stabilization ability of the guide RNA.  Then, Shibata and colleagues looked at how the stabilized Cas9–RNA complex targets DNA.  They confirmed that it binds to a pre-selected protospacer adjacent motif (PAM) site in the DNA.  A PAM is a short nucleotide sequence located next to the DNA’s target site, which is complementary to the guide RNA.

The research team’s high-speed movies further revealed that targeting (‘DNA interrogation’) is achieved through 3D diffusion of the Cas9–RNA complex.  Finally, the researchers managed to visualize the dynamics of the cleavage process itself: they observed how the region of ‘molecular scissors’ undergoes conformational fluctuations after Cas9–RNA locally unwinds the double-stranded DNA.

Article Title: Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy Journal: Nature Communications Authors: Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi, and Osamu Nureki

Doi: 10.1038/s41467-017-01466-8

Funders The Kao Foundation for Arts and Science, The Brain Science Foundation, JST/PRESTO, JST/CREST, The Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED, JSPS KAKENHI

[abbkine]

BioAdvisers said on Biotech Advisers

Visualization of CRISPR-Cas9 genetic-engineering technique

Researchers at Kanazawa University and the University of Tokyo report in Nature Communications the visualization of the dynamics of ‘molecular scissors’ — the main mechanism of the CRISPR-Cas9 genetic-engineering technique. This study provides unprecedented details about the functional dynamics of CRISPR-Cas9, and highlights the potential of HS-AFM to elucidate the action mechanisms of RNA-guided effector nucleases from distinct CRISPR-Cas systems.

CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.Selection of the site to be cut is done by a ‘guide RNA’ molecule bound to the Cas9 protein.  Now, a team of researchers led by Mikihiro Shibata from Kanazawa University and Osamu Nureki from the University of Tokyo has visualized the dynamics of the CRISPR-Cas9 complex, in particular how it cuts DNA, providing valuable insights into the CRISPR-Cas9-mediated DNA cleavage mechanism.

From left to right: Cas9 alone (apo-Cas9), Cas9 bound to RNA (Cas9–RNA), Cas9–RNA bound to its single-stranded DNA target (Cas9–RNA–DNA), Cas9–RNA bound to a partial DNA duplex (Cas9–RNA–DNA) and Cas9–RNA bound to its double-stranded DNA target (Cas9–RNA–DNA).

For their visualization studies, the scientists used high-speed atomic-force microscopy (HS-AFM), a method for imaging surfaces.  A surface is probed by moving a tiny cantilever over it; the force experienced by the probe can be converted into a height measure.  A scan of the whole surface then results in a height map of the sample.  The high-speed experimental set-up of Shibata and colleagues enabled extremely fast, repeated scans — convertible into movies — of the biomolecules taking part in the molecular scissoring action.

Fluctuations of the nuclease domain are indicated by magenta arrows. The cleavage products released from Cas9–RNA are indicated by blue arrows.

First, the scientists compared Cas9 without and with RNA attached (Cas9–RNA).  They found that the former was able to flexibly adopt various conformations, while the latter has a fixed, two-lobe structure, highlighting the conformational-stabilization ability of the guide RNA.  Then, Shibata and colleagues looked at how the stabilized Cas9–RNA complex targets DNA.  They confirmed that it binds to a pre-selected protospacer adjacent motif (PAM) site in the DNA.  A PAM is a short nucleotide sequence located next to the DNA’s target site, which is complementary to the guide RNA.

The research team’s high-speed movies further revealed that targeting (‘DNA interrogation’) is achieved through 3D diffusion of the Cas9–RNA complex.  Finally, the researchers managed to visualize the dynamics of the cleavage process itself: they observed how the region of ‘molecular scissors’ undergoes conformational fluctuations after Cas9–RNA locally unwinds the double-stranded DNA.

Article Title: Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy Journal: Nature Communications Authors: Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi, and Osamu Nureki

Doi: 10.1038/s41467-017-01466-8

Funders The Kao Foundation for Arts and Science, The Brain Science Foundation, JST/PRESTO, JST/CREST, The Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED, JSPS KAKENHI