2018/10/5

武田セミナー３10月6日

立体構造に基づいた
スーパーCas9
スーパーCas9の創出の創出

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)

1

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CRISPR-Cas9 system

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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
HNH

TargetDNA strand

TargetRepeat5’GuidecrRNA3’ (CRISPR RNA)

5’Anti-repeat

tracrRNA(trans-activating RNA)

PAM

3’(protospacer adjacent motif)
Jinek et al. Science20123

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Cas9 can work with a sgRNARuvC
Non-target
DNA strand
HNHTarget
DNA strand

TargetRepeatsgRNA

5’GuideAnti-repeat

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

Genome editing by Cas9Cong et al. Science
201342018/10/5

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

NHEJ

HDR

Repair

We can treat genetic diseases by cut & pasteof correct gene through genome editing
Ex vivo and in vivo genome editing treatment• Speedy and efficient introduction ofcorrect genes

Ex vivo

Extract self cells
Genome editing• Gene therapy by clean method• No problem of donor andtransplantation rejection because ofediting self cells

Plantation of correctedcells

In vivo

Intravenousinjection

Catheter

NATURE MEDICINE Vol21(2)2015
105

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Nureki Nishimasu Feng

Our collaboration started
Since 2009.

First crystal structure of Cas9 complex

Nishimasu et al. Cell (2014) Anders et al. Nature 2014 (Jul)62018/10/5

R:AR duplex

REC(Repeat:anti-repeatduplex)

HNH

PI (PAM-interacting)

RuvC
Collaboration with Feng Zhang

Nishimasu et al. Cell 2014

Guide RNA:Target DNA heteroduplex
TargetGuide
Stem loop 1

5’Repeat5’

Tetraloop3’

Anti-repeat

Stem loop 2

Stem loop 3
3’

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Guide RNA:Target DNA recognition

Guide RNA:Target DNA recognition8

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Guide (seed region) recognition

Cas9–sgRNA–DNA complex

9

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Phosphate lock loop facilitates DNA unwinding

NUC (Nuclease)
lobeREC (Recognition)
lobe

SaCas9SpCas9

Guide:target
heteroduplex

PAM

RuvCPI (PAM-interacting)Nishimasu et al. Cell 201410

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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

Arg1335
Arg1333

PAM (NGG) PI domain
Anders et al. Nature 2014 (Jul)112018/10/5

Cas9-apo Cas9-sgRNA
ClosedOpen

Jinek et al. Science 2014

Jiang et al. Science 201512

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Hi--speed AFM
Hi

Shibata, Nishimasu et al., Nat. Commun. (2017)

Shibata, Nishimasu et al., Nat. Commun. (2017)13

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Weakness of SpCas9
for genome editing
 SpCas9 is large (1368 aa)
 Low efficiency of packaging into viral
vectors for delivery to somatic tissues

 SpCas9 requires the NGG
PAM Limited target selection

MiniCas914

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SaCas9 is Small> 300 aa
17% idRan et al. Nature 2015

Different PAM
NNGRRT (R = A/G)
SaCas9?
GGT

NGG
SpCas9Arg1333

Arg133
5(Anders et al. 2014 Nature)15

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Different sgRNAGuide

R:AR duplex

SL1SaCas9

SL2Guide

R:AR duplex

SL1SpCas9SL3SL2Overall structure

Nishimasu et al. Cell (2015)16

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NNGRRT PAM
TTGAAT PAM complex (2.6 Å)
TTGGGT PAM complex (2.8 Å)

Asn9855’-NNGRRT-3’
Glu993

Arg1015

Arg991

Asn985/Asn986/Arg991

Collaboration with Feng Zhang lab

Helicase activity of Cas9 is important for DSB.

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SaCas9SpCas9
R:AR duplex

REC

R:AR duplex

REC

WED
WEDRuvC

PIRuvC

Nishimasu et al. Cell 2015

SaCas9Anders et al. Nature 2014

SpCas9
R:AR duplex

RECPI

R:AR duplexREC

WEDWEDRuvC

PI

Nishimasu et al. Cell 2015

RuvC

PI

Anders et al. Nature 201418

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SpCas9 SaCas9 CjCas9
Streptococcus pyogenes

Staphylococcus aureus

Campylobacter jejuni

II-A

II-A

II-C

1368 aa 1053 aa 984 aa
NGG

NNGRRTNNNNACA?

Fonfara et al. Nucleic Acid Res 2013
Ran et al. Nature 2015

Mininal functional unit of Cas9

HNH domain omitted19

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Yamada, Watanabe et al. Mol Cell 2017

PAM recognition
NNNVRYACTS
PAMNTS

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But CjCas9 has weak in vivo activity…

Only two papers have been reported for in vivo
genome editing by CjCas9.

New mutant

PatentedWT

21

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PAM simplification

New technology using Cas9-sgRNA system
Deaminase

Base editing

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Cas9 variant that recognizes a simple PAM• allows you to target more sites in thegenome. You can more freely design thesgRNA, which may prevent the recentlyreported risk of large DNA deletion ingenome editing• but also allows you to more precisely
perform base editing• and also allows you to more efficiently
perform transcriptional regulation
depending on the sgRNA design

Cas enzyme

PAM

SpCas9
SaCas9
AsCas12aNGG
NNGRRTTTTV

SpCas9 VQR
SpCas9 VRER
AsCas12a RVR
AsCas12a RR

NGA
NGCG
TWTV
TYCVRan et al. Nature 2015, Zetsche et al. Cell 2015
Kleinstiver et al. Nature 2015, Gao et al. Nat Biotechnol 201723

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Rational Engineeringof Cas9

NGG

NG

Since 2015

RECG2

RNA

HNH

G3

R1333
DNA

E1219
R1335

RuvC

PI

Anders et al. Nature 201424

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NGG
NNCGNGC
GR1333 R1335

NNC
GArg Arg

CGR1333 R1335
A25

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in vitro cleavage
1. WT SpCas9
2. R1335A
3. R1335A + Mut 1
4. R1335A + Mut 2
5. R1335A + Mut 3…
in vitro cleavage (TGG)

IVC (200 nM Cas9-RNA, 37C, 30 min)Microchip electrophoresis

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in vitro cleavage (TGG)
WT SpCas9

ARRRR

D1135V (VQR)

G2
R1333

E1219F (model)
R1335V (model)

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in vitro cleavage (TGG)
WT SpCas9

VRRRR + D1135V/E1219F
ARRRR + D1135V/E1219F
ARRRR + D1135V
ARRRR

in vitro cleavage (TGN)282018/10/5

WT SpCas9

SpCas9-NG

WT SpCas9

SpCas9-NG

In collaboration w/ Zhang lab @ BroadInstitute29

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Crystal structure of SpCas9-NG
L1111R
D1135V
G2G3

A1322R
E1219F
R1333 R1335V

G1218R

T1337R

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xCas9 (NGPAM)
A262T/R324L/S409I/E480K/E543D/M649I/E1219V

xCas9312018/10/5

WT SpCas9

G2

R1333

G3

E1219VR1335

1. WT SpCas9
2. SpCas9-NG
3. xCas932

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in vitro cleavage (TGN)WT SpCas9 SpCas9-NG

xCas950 nM Cas9-sgRNA332018/10/5

WT SpCas9 SpCas9-NG

xCas9

200 nM Cas9-sgRNA

Activity in HEK293T

In collaboration w/ Zhang lab @ BroadInstitute34

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Activity in HEK293T

In collaboration w/ Zhang lab @ Broad
Institute

C to T base editing
Target-AID
SpCas9/SpCas9-NG D10A nickase

AID

PmCDA1 cytidine
deaminaseNishida et al. Science 201635

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C to T base editing

Mutation frequency (%)

Poly-CIn collaboration w/ Yachie lab @ U Tokyo

C to T base editing

Mutation frequency (%)

Poly-CIn collaboration w/ Yachie lab @ U Tokyo36

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Poster 072

Brevibacillus laterosporus Cas9•

Type II-C Cas9 endonuclease• Small size (1092 a.a.)
CjCas9 < SaCas9 ≈ BlCas9 < SpCas9•Simple PAM recognition
5′-NNNNCNDD-3′ PAM(N = A or T or G or C, D = A or G or T)•170 mer sgRNA•

Genome editing in plant (maize)(Karvelis et al. Genome Biology ,2015)

in vitro cleavage assay
PAM preference

CCGAC

CCGAG

CCGAT

CCGCA

CCGGA

CCGTA

CCTAA

CCCAA

CCAAA

CTGAA

CGGAA

CAGAA

GCGAA

TCGAA

ACGAA

CCGAA

5′-TTTNNNNN-3′Cut (2 kb)

SubstrateProductProduct37

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Comparison between Cas9 Orthologs
1009080

Cleavage (%)

70

SpCas96050

SaCas94030

CjCas92010BlCas90012345Time (min)Unpublished

Crystallization and Structure Determination
SPring-8

BL41XU

200 µm

0.2 M Ammonium sulfate
0.2 M Sodium acetate pH4.5
20% w/v PEG500 MME
10 mM strontium chloride

2.4 Å

Molecular Replacement (CjCas9)

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Overall Structure
REC lobe
45

177

RuvC-I BH

230

REC1454

REC2504

671

807

821923

RuvC-II -(GGGSGG)- RuvC-III Linker

WED1092PI

NUC lobe

REC2REC1

REC lobe

BH

Guide RNANUC lobe

WEDPAM

Unpublished

DNA
RuvC

2.4 Å Resolution
Rwork/Rfree = 0.22 /0.27

PI

Structure of sgRNAC80

G71
C103Repeat
G
5’ GGAAAUUAGGUGCGCUUGGC GCUAUAGUUCCUU A
Tetraroop
Guide Sequence
UGAUAUCGUUGAA A

sgRNAAC

Stem Loop2 U
CCCCA
UUCC
GCUA
GGGG

GGC
GU

A Anti-repeat
A G
G G
C G
C C
C
A A
G C
A C

Stem Loop 1A

A78
U72
U104

U3’39

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Structure-guided engineering (activation)

UnpublishedPreference of 7th and 8th DD

系列25min

TTTCCGNNWT BlCas9100

系列110minmutant BlCas99080706050403020100AACA

ACCCAACAAC

CC

Unpublished40

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PAM Recognition Mechanism
5′-NNNNCNDD-3′ PAM

C5*
Asp1022

Lys1040G(-5)

D1022/K1040 – C:G PAM

Cleavage rate
10.8
0.6
0.4
0.20

WT

D1023ND1022N

Unpublished

PAM Recognition Mechanism

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Structure-guided engineering (PAM)
Asp10225’ NNNNC
3’ NNNNG

3’ NTS
5’ TS

WT
NNNNC PAMWT
TTTCN PAMD1022N

C A T G C A T G

Lys1040
D1022N
Asn10225’ NNNNA
3’ NNNNT

3’ NTS
D1022N
5’ TS NNNNA PAM ?

Recognizes NNNND PAM unexpectedly

Lys1040

Unpublished

Combination of WT and D1022N mutant
technically generated PAM-less Cas9WT

5’TTTCN

C A

5min

D1022NT G C AWTT G C A

10min

D1022NT G C T AGPATENTEDCA T G

84

D1022N changes 5th PAM preference from C to D (A , G , T)42

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Length of Guide Sequence
0.5min
系列21min系列3系列12min

1009020bpsgRNATarget gene

22bp

sgRNA

Target gene

24bp

sgRNA

Cleavage activity (%)

Target gene

8070605040302010020bp

21bp

22bp

23bp

24bp

Generation of “Super Cas9” by resolving three big
problems by structure-based CRISPR engineering
Off target
cleavageHigh fidelity

Cleavage limit by
PAM recognition

More freely

SuperCas9
smaller
Low introduction
efficiency8643

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Acknowledgements

Hiroshi Nishimasu
Seiichi Hirano
Toshihiro Nakane@U Tokyo
Tsukasa Ohmori
Yutaka Hanazono@Jichi Med. Univ.

Feng Zhang
Omar Abudayyeh
Jonathan Gootenberg
F. Ann Ran@MITFunding
BL32XU, BL41XU
beamline staff@SPring-844