Epigenome editing ; application ofgenome editing tools

2016.06.09
M1 Yamaji Kyohei
1Contents

1. Genome Targeting in Catalysis Medicine
2. Genome Editing Tools
1. Introduction
2. ZFN
3. TALEN
4. CRISPR/Cas9
3. Epigenome Editing
4. Summary2

Epigenetics ; chemical modifications
DNA/Histone modifications can control its gene expression patterns.
DNA methylation pattern is different between stem cells and differentiated cells

stem cells

Differentiated cells

methylation

Dirk Schübeler
Nature 462, 296-297

differentiatedundifferentiatedExpression of some genes is suppressed in differentiated cells with reduced
global Histone acetylation.
Expression ofDifferentiated cells
gene A and B

Histone proteinsDNAstem cells

Expression ofgene A and B 3
Loring, J F, et al. Cell Research (2014) 24:143–160

4

Cataｌysis medicine
In some cancer cells, tumor suppressor genes are suppressedwith decreased Histone acetylation.
Inducing Histone acetylation can be a hopeful anti-cancer strategy.

tumor suppressor gene(e.g., p53)

anti-cancer
agentstumor suppressor gene
Manel Esteller (2007)

5

Cataysis medicine
Mutations of HAT are frequently found in B-cell lymphoma.

CBP(HAT)

Inactivating mutations of acetyltransferase genes in B-cell lymphoma
Pasqualucci, L. et al. Nature 2011, 471, 189.

HAT-independent Histone acetylation can be the solution?

6Cataysis medicine
Histone acetylation level is regulated on the balance of two
catalysts called HAT and HDAC.
What if HAT is defected...?
HAT (histone acetyltransferase)+ Acetyl-CoA (donor)

Ac
Ac

Ac
Ac

HDAC (histone deacetylase)
OHNO

NHOH

SAHA/Vorinostat/Zolinza

HDAC inhibitor is actually used as an anti-cancer drug.

HAT-independent histone acetylationby artificial catalyst system
Artificial Catalyst System
for histone acetylation
NEWdrug?

AcAc

Ac
Ac

HDAC (histone deacetylase)transcriptionOFF

transcription
ONSAHA/Vorinostat/Zolinza
7

Current catalysis medicine
SynCAc acetylate genome-wide histones nonspecifically
The result of In nucleo ChIP (No compound vs SynCAc) by Dr.Ishiguro•HeLa nuclei were acetylated with 50µM 8DMAP, 10µM 3NMD-8R @25℃ for 3hrs in SynCAc.
(8DMAP : batch-C by Dr. Amamoto, 3MMD-8R : KY2457 by Dr.Yamatsugu)

Chr1 (heterochromatin)
120%
100%80%H3K4me3
H3K9me3
H3K18Ac

Chr4 (heterochromatin)
100%
80%

H3K4me3
H3K9me3
H3K18Ac

Rhob(euchromatin)
100%
80%

H3K4me3
H3K9me3
H3K18AcGAPDH(euchromatin)
100%
80%

60%

60%

60%

40%

40%

40%

20%

20%

20%

0%0%0%

H3K4me3
H3K9me3
H3K18Ac

60%
40%
20%
0%

Both hetero/euchromatin are targets of SynCAc.
(heterochromatin acetylation might be stronger)

Without targeting, genome-wide histone acetylation is observed
8Gene targeting and catalysis medicine
The usefulness of Gene targeting of artificial catalyst
・ medical application
・ applications as biological tools
Applications of artificial catalyst as biological tools
・ roles of various acylations on histone
・ roles of modifications on various residues of histone→internal controls are easily obtained in targeting of catalyst
For further applications…
・ genome-wide screening (acetylation)
And etc…
Gene targeting can expand the possibility of artificial catalyst.
9

Contents

1. Genome Targeting in Catalysis Medicine
2. Genome Editing Tools
1. Introduction
2. ZFN
3. TALEN
4. CRISPR/Cas9
3. Epigenome Editing
4. Summary
10

The utility of Genome editing
Genome editing
Genetic engineering in which DNA is inserted, deleted, or replaced in the genome of anorganism using engineered nucleases, or "molecular scissors.“ (wikipedia)
General procedures for producing
knockout/knockin mice
The 2007 Nobel Prize in Physiology or Medicine“principles for introducing specific genemodifications by the use of embryonic stem
cells” (established : 1989)“the beginning of new era in genetics”
However…
Takes so much time and trouble

Genome editing tools havemade this drastically easier
11Genome editing tools
1st generation : ZFN (zinc finger nuclease)
ZF modules : protein to bind DNA
Nuclease : protein to cleave DNA

2nd generation : TALEN (transcription-activator-like effector nuclease)
Nuclease : protein to cleave DNA
TALE : protein to bind DNA

3rd generation : CRISPR/Cas9
Cas9 :protein to bind and cleave DNA

gRNA : RNA to bind DNA

Genome editing tools…
recognize specific sequencescleave DNA strand

実験医学別冊 今すぐ始めるゲノム編集 TALEN＆CRISPR.Cas9の必須知識と実験プロトコール 山本卓編

12

Principle of genome editing
genome editing tools promote DNA repair dependent mutations
to specific DNA regions
Scheme for the specific gene knockout or knockin by genome editing tools
Genome editing tools introduce
DSB to specific DNA region

Two intrinsic pathways
for DNA repair
Ligation(error-prone)

Use another homologous
DNA strand as a template

Homologous region

Homologous region

some bases are often
lost in repair
13

https://www.cosmobio.co.jp/support/technology/a/crispr-talen.asp

Contents

1. Genome Targeting in Catalysis Medicine
2. Genome Editing Tools
1. Introduction
2. ZFN
3. TALEN
4. CRISPR/Cas93. Epigenome Editing
4. Summary14

Fok1 endonuclease
ZFN consists of Fok1 endonuclease cleavage domain and ZF protein.
Fok1

ZF protein

ZFN needs to be designed to put the cleavage
site in between

Fok1 endonuclease consists of Recognition domain(RD) and Cleavage
Recognition domain binds 5’-GGATG-3’
domain (CD)(natural Fok1 is sequence specific)

Cleavage domain dimerization is necessary forthe cleavage
5’-GGATG-3’
RD1
CD1
DNA1
CD2

RD2

DNA25’-GGATG-3’
Aggarwal AK., et al., Nature. 1997
Jul 3;388(6637):97-100.

Aggarwal AK., et al., J. Mol. Biol. (2001) 309, 69±7815

structure of zinc finger protein
ZF motif contains Zn, which interacts with His2Cys2 and stabilizes ββαStructure of finger 2 from Zif286, a kind of ZF motifs
His
Cys

fingerHis

Zn2+Cys

F or Y

3-5 aminoacids

1232 or 4amino acidshydrophobic

Potential DNA binding residues(numbered from the start of α helix)

Residues closest to DNA binds DNA(distance depends on the structure of ZF)
Pabo CO., et al., Annu. Rev. Biophys. Biomol. Struct. 2001. 70:313–4016

DNA recognition by zinc finger protein
How ZF motifs recognize DNA
ZF motifs interact withDNA major groove

One residue recognizes
one nucleobase
Arginine-guanine
Asparagine-adenine

For sufficient sequence specificity,
3-6 motifs are usually combined.

Pabo CO., et al., Annu. Rev. Biophys. Biomol. Struct. 1999. 3:183–212
Pabo CO., et al., Annu. Rev. Biophys. Biomol. Struct. 2001. 70:313–40
17

Stabilizing residues of zinc finger-DNA complex
Other interactions between ZF motifs and DNA
a. His (Nd)-phosphate of the DNA backbone (5’- primary strand)b. Lys or Arg-phosphate of the DNA backbone (the same phosphate as a. or its 3’strand)c. Lys in the linker (TGEKP)-phosphate of the DNA backbone (5’- primary strand)

The linker (TGEKP) contributes to the stability of ZF-DNA complex
C-cap
T : make hydrogen bond withNH in the next Helix (C-cap)
G : complete the C-cap
E : stabilize finger-finger
interaction (contact R ofHXRXH in the next finger)
K : contact DNA backbone
P : p-p stacking with -1 residue
in the next finger

HisCysZn2+His

LysCysorArg

18

Rose GD., et al.,Helix capping.Protein Sci. 1998, 7:21-38

Drawback of zinc finger protein
drawback of ZFNAssembled motifs often fail to recognize predicted sequence
Predicted sequence : GGA-CCA-AAA-AGA-GAT-GTA
Primary strand
Complementarystrandactual sequence : GGC-CCG-AAA-AGG-GAT-GTA
Weak specificity
Primary strand

ComplementarystrandUnexpalained specificity, changed interaction by water, changed interaction byglycerol(gol, necessary for crystallization, substitution of water?)

Horton, NC. et al., J. Mol. Biol. (2006) 363, 405–421

19Structural insight behind the DNA recognition by ZF
reason for drawback of ZFNThe broad range of variation in the docking arrangement of ZF-DNA complex
Finger1-3 of Zif268
Other Zif-like ZF① ②DNA strand

Pabo CO., et al., Annu. Rev. Biophys. Biomol. Struct. 2001. 70:313–40

The change in docking arrangement
sometimes leads to the loss of baserecognition by some residues
Modular assembly may change thedocking arrangement
20

Contents

1. Genome Targeting in Catalysis Medicine
2. Genome Editing Tools
1. Introduction
2. ZFN
3. TALEN
4. CRISPR/Cas9
3. Epigenome Editing
4. Summary
21Overlook on TALENTALEN consists of Fok1 nuclease domain and TALE (DNA binding protein)
Fok1 Nuclease : DNA cleavage

DNA binding modules
D. A. Wright et al., Biochem. J., 462, 15-24 (2014)

TALEN needs to be designed to put the cleavage site in between like ZFNTALEN has overcome ZFN in terms of its easiness in designing andspecificity
Predicted sequence is (almost) always recognized. (drawback of ZFN is solved)
Only restraint is that the target sequence has to start from thymineHigh specificity (many more bases can be recognized than the other two tools)

Easy to design, usable to various sequences, and high accuracy oftargeting
22

TALE in natural world
TALE (Transcription-activator like effector) in plant infectious bacteria
TALEbacteria

Schematic view of domains in TALE
Nuclearlocalization

Transportation
Transportation DNA binding

activation

Plant cell
TALE in application as a genome
editing tool
Nuclear
Bacteria activates specificgene by TALE andfacilitates its growth

localization

Transportation DNA binding

activation

Bonas U. et al., Curr Opin Microbiol. 2009 Feb;12(1):37-43.
23

Structure of TALEN
Mechanisms for DNA binding

Schematic view of TALE sequence

RVD : repeat-variable diresidue
Van Der Waals
interaction‘HD’ recognizes Cytosine
Van Der Waals
interaction

N-terminus

The repeat (0, -1)
recognizes Thymine

Interactions between TALE
and DNA major groove

C-terminus

A. N. Mak et al., Science, 335, 716-719 (2012)

Hydrogen bondwith N424

DNA sequence recognition by TALE
RVDs and their specificity

Interact with base

Interaction between ‘NG’ and Thymine

VDW between a-Carbon of Gly
and methyl group of thymine

Stabilize TALE

Bogdanove AJ., et al., Science. 2009 Dec 11;326(5959):1501.

Interaction between ‘NN’and Guanine

Interaction between ‘NN’and Thymine

Hydrogen bond with N7
nitrogen of Guanine or Adenine(both purine ring)

VDW between a-Carbon of Glyand methyl group of thymine

N or H (the 1st of RVD)
stabilize the structure

H-bond withcarbonyl oxygenof residue 8 of thesame repeat

A. N. Mak et al., Science, 335, 716-719 (2012)25

Contents

1. Genome Targeting in Catalysis Medicine
2. Genome Editing Tools
1. Introduction
2. ZFN
3. TALEN
4. CRISPR/Cas9
3. Epigenome Editing4. Summary26

Overlook on CRISPR/Cas9
CRISPR/Cas9 consists of Cas9 protein and gRNAsgRNA : optimized gRNA

TargetDNA

Cas9 cleaves DNA strand

Essential

Cas9gRNA(guideRNA)

EnhanceEnhancestability
stability
Nishimatsu, H., et al. (2014) Cell, 156, 935-49

Residues responsible for DNA cleavage

Sequence recognition
depends on gRNA
Jinek, M., et al. (2012) Science, 337, 816-21

DNA recognition depends on RNA, easily modified
restraint of PAM sequence(only 3~6 base)Multiplex editing

Cleaved DNA

Little cost and effort for design

27Jinek, M., et al. (2012) Science, 337, 816-21

CRISPR/Cas9 in natural world
CRISPR/Cas is originally an immune system against nucleic acids from viruses
1. Immunization
Nucleic acids from viruses are cleaved
and inserted into CRISPR locus

Activate gRNA

Cas : Cleave DNA

DNA Sequence fromviruses are stored

2. Immunity

Cas9 cleaves invading DNA from virus
recognizing the complementary
sequence to the stored one.
To distinguish unself (virus) and self,
Cas9 recognizes preceding PAM
sequence.
Activate gRNA

Mali, P., et al., Nature Methods, 2013, 10, 957-62Cas : Cleave DNA

DNA Sequence from28
viruses are stored

Design and structure of CRISPR/Cas9
Restraint of PAM sequence is
not a problem for practical usesgRNA; DNA; Cas9 ternary complex

Jacks, T., et al., Nat Rev Cancer. 2015 Jul;15(7):387-95.

gRNA must include PAM (NGG) but often
several can be designed for 1 gene
Nishimatsu, H., et al. (2014) Cell, 156, 935-49

positively charged groove of CRISPR/Cas9interacts with negatively charged sgRNA;
DNA complex
Cong, L., et al. (2013) science, 339, 819-22

See appendix for detailed interactions29

Specificity of CRISPR/Cas9
Drawback? of CRISPR/Cas9

Strategies to improve specificity ofTALEN is often better than CRISPR/Cas9 in Specificity CRISPR/Cas9
1. Truncate sgRNA target sequence by 2
Number of off-target sites of CRISPR/Cas9
or 3 nucleotides (bases which weakly
recognize target sequence)
2. two Cas9s for one target gene.
3. Cas9 mutants
2-1. Cas9-Fok1
Fok1 : cleave DNA

Kuscu, C., et al. (2014) nature, 32, 677-83

Joung JK., et al. Nat
Biotechnol. 2014
Jun;32(6):569-76

Depending on sgRNA, 10-1,000 off-target sites exist.

CRISPR/Cas9 and TALEN are not so different interms of mismatch tolerance per recognition bases

Cas9 mutant with no DNA
cleavage ability

2-2. Cas9 nickase (cleave only
one of the DNA double strand)

CRISPR/Cas9 : 1-3 mismatches / 20 bases
TALEN : 1-2 mismatches / 18 repeats
Mali, P., et al. (2013) nature biotechnology, 31, 833-38

Ran, F. A. et al., Cell 154, 1380–1389 (2013).

30

CRISPR/Cas9 mutant with high specificiyConstruction of highly specific Cas9 mutantProblems about former attempts to increase specificity
・ unproven efficacies on genome-wide scale
・ potential to create more new off-target
・ need for fusion of functional domains to Cas9・ reduction in targeting range
・ more challenges for delivery

Necessity for the development ofa robust and easily used strategy

Mutations to reduce Cas9-DNA interactions Mutated to Alalead to the increase in specificity

MutantJoung JK., et al., Nature. 2016 Jan 28;529(7587):490-5

31

Contents

1. Genome Targeting in Catalysis Medicine
2. Genome Editing Tools
1. Introduction
2. ZFN
3. TALEN
4. CRISPR/Cas9
3. Epigenome Editing
4. Summary32

Epigenome editing tools
two ways to deliver effectors to specific DNA locus
1. Directly fuse effector domains to DNA binding protein
LSD1 : histone demethylase

Bernstein BE., et al., Nat Biotechnol., 2013 Dec;31(12):1133-6.

2. Use of RNA binding protein

interaction

Konermann, S. et al., Nature, 2015, 517, 583-833

Use of histone demethylase fused TALE
two ways to deliver effectors to specific DNA locus
1. Directly fuse effector domains to DNA binding protein
LSD1 : histone demethylase
LSD1 catalyzed the removal of H3K4 methylation
around targeted region

decrease

Quantitative dataAlso, removal of H3K27ac was observed(by the recruitment of HDAC)

decreasedecrease
34

Epigenetics study using TALE-LSD1①
Prior study indicate that sequence elements enriched for H3K27ac
and H3K4me2 exhibit enhancer activity
TALEN-LSD1 enables post-translational modifications to be artificiallyintroduced and their effects to be evaluated.
Four of nine enhancers tested, nearby genes were inactivatedReproduction
experiment

Why did the other show little change?

Weak transcriptional effect under detection threshold?
No function as an enhancer in the cell tested?

35

Epigenetics study using TALE-LSD1②
In the same enhancer, the region where TALEN-LSD1 is targeted maychange the degree of effectThree enhancers for ZFPM2 were tested, yielding different inactivation of gene
expression

About 2-fold
reduction

Only 10-20%reduction
Not statisticallysignificant

Effect of targeting paired enhancers is lessthan the sum of their individual effects
Redundant effect of the multiple enhancers

TALE-LSD1 fusions are valid tools to study complex regulatory interactions
among multiple enhancers and genes in a locus

36Specific gene activation tool using CRISPR/Cas9
two ways to deliver effectors to specific DNA locus
1. Directly fuse effector domains to DNA binding protein

LSD1 : histone demethylase

2. Use of RNA binding protein

interaction

37

Structure based design of SAM system
Structure based design for construction of RNA aptamer
Tetraloop and stem loop2 is exposed
outside Cas9
Hairpin aptamer
Hairpin aptamer binding protein
bindTranscription
factors

Good environment foreffector proteins to function→appropriate for scaffold for
effector proteins38Activation of specific gene achieved with SAM
Confirmation of the validity of the design
Efficient gene activation was achieved dependent on the numberof hairpin aptamers

Structural position of hairpin aptamers is important

Little/no activation

Not exposed on the surface of Cas939

Contents

1. Genome Targeting in Catalysis Medicine
2. Genome Editing Tools
1. Introduction
2. ZFN
3. TALEN
4. CRISPR/Cas9
3. Epigenome Editing
4. Summary40

Summary・genome editing tools facilitated the study of gene functions・ZFN was the first to be invented but has not been widely used due
to its difficulty in designing
・ TALEN overcame the drawback of ZFN due to its simplicity inrecognizing DNA sequence.
・CRISPR/Cas9, using gRNA for recognition of DNA sequence,
rendered genome editing much easier and is now intensively studied
・genome editing tools are applicable to edit epigenome, enabling
more direct study of epigenetics.
41

Appendix; detailed interactions of Cas9-gRNA-DNA

42

ZFN (Zinc-finger nuclease)
ZFN consists of Fok1 endonuclease cleavage domain and ZF protein.
Fok1 endonuclease consists of Recognition domain and Cleavage domain
Recognition domain binds 5’-GGATG-3’

43

Cole P A, et al. Chem. Rev., 2015, 115 (6), pp 2419–2452

TALEN (transcription-activator-like effector nuclease)
RVDs and their specificity

Structure of the DNA
binding repeat‘N*’ extends less deeply
into the major groove

44

CRISPR/Cas9
Application to genome-scale gene activation screen45