2018/8/20

武田セミナー2018年8月18日

クリスパーCas9

濡木 理
東京大学大学院理学系研究科

Nucleotide sequence of the iap gene, responsible for alkaline
phosphatase isozyme conversion in Escherichia coli, andIdentification of the gene product.
Y. Ishino et al. J. Bacteriol. (1987)

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CRISPR-Cas9 system

Cas9 is a putative nuclease
Streptococcus pyogenes Cas9 (SpCas9)11368?

?

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Cas9 is an dual RNA-guided DNA nuclease

Jinek et al. Science2012

Cas9 is an dual RNA-guided DNA nuclease
RuvC
Non-target
DNA strand
HNHTarget
DNA strand

TargetRepeat
5’GuidePAM

crRNA
3’ (CRISPR RNA)

5’Anti-repeat

tracrRNA(trans-activating RNA)3’(protospacer adjacent motif)
Jinek et al. Science20123

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Cas9 can work with a sgRNA
RuvC
Non-target
DNA strand
HNH

TargetDNA strand

TargetRepeatsgRNA

5’GuideAnti-repeat

PAM(single-guide RNA)3’(protospacer adjacent motif)
Jinek et al. Science
2012

Genome editing by Cas9Cong et al. Science20134

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Genome editing by Cas9

Charpentier & Doudna Nature N&V2013• Introduction of Cas9 and 5sgRNAs into mouse ES cells
enabled simultaneous knockout of 5 genes.
• Two gene knocked-out
mouse was generated byCas9.

Wang et al. Cell 20135

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Double strand break in genome causes non-homologous
end joining (NHEJ) or homologous recombination (HDR)

ゲノム編集は決まった位置に切れ目を入れるだけ、あとは細胞が自然の摂理で修復する過程を遺伝子矯正に利用するのみ

NHEJ

HDR

RepairCRISPR(ゲノム編集)は遺伝子治療法開発にブレークスルーをもたらす強力な技術•

体外に細胞を取り出して編集する方法と体内で編集する方法がある

体外および体内でのゲノム編集医療
体外
自己細胞採取ゲノム編集•

短時間かつ効率のよい設計と導入が可能になる•痕跡のないクリーンな方法によって遺伝子を修復できる•

自己の細胞を編集するので探すのが大変な移植ドナーが不要正常化した自己細胞の注射
体内

全身投与

局所投与

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Question
How does Cas9–sgRNArecognize and cleave DNAtargets?

Nureki Nishimasu Feng

Our collaboration started
Since 2009.

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SpCas9–sgRNA–DNA complex

SpCas9–sgRNA–DNA complex

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Crystallization by vapor diffusion
Hanging drop法

Sitting drop法

自動結晶化装置

Example: crystallization of glutamyl-tRNA synthetase92018/8/20

Fourier transformation

X-ray crystallography

X-ray

Preparation of Cas9-sgRNA-DNA complex
Cas9

sgRN
ACas9
(160 kDa)

Cas9-sgRNA complexCas9-sgRNA-DNA complex(185 kDa)(190 kDa)

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Crystal ofCas9–sgRNA–DNA
0.3 mmData collection

2.8 Å

BL32XU @ 2013/11/17
Native1.000 Å2.5 Å res (I/sigma = 2.1)

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2.5-Å resolution crystal structure
GuideRepeat

TargetAnti-repeat

Stem loop 1

First crystal structure of Cas9 complex

Nishimasu et al. Cell (2014)12

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HNH

NUC (Nuclease)lobe

REC (Recognition)
lobeRuv
CRE
C

R:AR duplex(Repeat:anti-repeat
duplex)PI (PAMinteracting)Nishimasu et al. Cell
2014

Guide:target
heteroduplex

PAMRuv
特許出願 CPI(PAMinteracting)Nishimasu et al. Cell
2014

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Guide RNA:Target DNA heteroduplex
TargetGuide
Stem loop 1

5’Repeat5’

Tetraloop3’

Anti-repeat

Stem loop 2

Stem loop 3
3’

Guide RNA:Target DNA recognition14

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Guide RNA:Target DNA recognition

Guide (seed region) recognition15

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Guide:Target recognition

Three-way junction

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Repeat recognition

Anti-repeat recognition17

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Stem loop 1 recognition

Stem loops 2 & 3 recognition18

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Cas9 is an dual RNA-guided DNA nuclease
RuvC
Non-target
DNA strandHNH

TargetDNA strand

TargetRepeat5’GuidePAM

crRNA
3’ (CRISPR RNA)

5’Anti-repeat

tracrRNA(trans-activating RNA)3’(protospacer adjacent motif)
Jinek et al. Science2012

New nickase

RuvC domain

HNH domain

Nishimasu et al. Cell 2014、patent PCT/US2014/069925

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PAM-interacting domain

Martin Jinek @ Univ. of ZurichAnders et al. Nature 2014 (Jul)20

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SpCas9–sgRNA–target DNA

SpCas9–sgRNA–target DNA+ non-target DNA (PAM)

PAM (NGG)
Anders et al. Nature 2014 (Jul)Nishimasu et al. Cell 2014 (Feb)PAM is recognized by the PI domain
GGT

Arg133
3Arg133
5

PAM (NGG) PIdomain

Anders et al. Nature 2014 (Jul)212018/8/20

Jun 28, 2012
Jinek et al. Science
Biochemical characterization of Cas9
Feb 6, 2014
Jinek et al. Science
Apo Cas9

Feb 13, 2014
Nishimasu et al. Cell
Ternary complex

Jennifer Doudna Martin Jinek

Jul 27, 2014
Anders et al. Nature
Quaternary complex

Osamu Nureki Feng Zhang

Martin Jinek

Cas9-apo Cas9-sgRNA
ClosedOpen

Jinek et al. Science 2014

Jiang et al. Science 201522

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Hi-speed AFM

Poster 15

Shibata, Nishimasu et al. submitted23

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Shibata, Nishimasu et al. inpreparation24

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Cas9-sgRNAを応用した新規技術「切らないCRISPR」技術は遺伝子をピンポイントでオン・オフできる
CRISPR-GNDM (Guide Nucleotide-Directed Modulation) システム

遺伝子調節タンパク部位特異的
ヒストン修飾

転写活性化/抑制化
ヒストン脱/メチル化・アセチル化

遺伝子ON/OFFAc

Me

ガイドRNA
ターゲット遺伝子mini-dCas9

わざと切れなくした
小型改変CRISPR酵素gRNA: guide RNA=ゲノム上の特定の位置をポイントする

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Structure-guided design of SAM complex
Tetraloop
MS2-binding aptamer

Stem loop 2

Synergistic activation mediator (SAM)
1. dCas9 (D10A/N863A) + VP64
2. sgRNA + MS2-binding aptamer
3. Bacteriophage MS2 coat protein+ P65 + HSF1 (human heat shockfactor)
VP64, P65, HSF1: transcription activator

Konermann, Brigham et al. Nature 201426

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Gene activation by SAM

Gene activation by SAM

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Multiplexed gene activation

Genome-scale gene activation screenA375
melanoma cells

PLX-4720
BRAF inhibitor

70,290 sgRNAs(3 for 23,430 genes)

Gain-of-function changes that confer PLX-4720 resistance
BRAF Thr/Ser kinase V600E mutation -> A375 melanoma cells

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sgRNA enrichment after PLX-4720 treatment

特許出願29