1© 2017 Editas Medicine© 2017 Editas Medicineeditasmedicine.com
Defining and characterizing the components of a CRISPR-Cas9
genomic medicineHari Jayaram
CRISPR International Conference 2017June 10, 2017
2© 2017 Editas Medicine
Presentation Overview
Sickle Cell Disease, b-globin Gene Regulation, and Editing Strategy
RNP based Lead Discovery platform
Fully synthetic modular dgRNA
Ex-vivo T-cell and HSC editing with Cpf1
Platform for CRISPR medicines
3© 2017 Editas Medicine
Pipeline strategy to enable successful medicines
4© 2017 Editas Medicine
Scalable, Consistent Engineered Cell Therapies
Engineer multiple components to T-cell & HSC sensitivity (maintain cell viability, potency)
Platform for CRISPR Medicines
EngineeredPatient Cell
PatientCell
RNA GuidedEndonuclease
RNAGuide
Ribonucleoprotein Particle (RNP)
+
Electroporation
5© 2017 Editas Medicine
Aspects of a Ribonucleoprotein medicine
Defining every component : key to a successful CRISPR medicine
Guide Sequence
+ =
• Efficacy: well optimized cellular and well characterized in vitro efficacy
• Stability: track RNP activity through gene-editing workflow and therapeutic window
• Fidelity: Define every component and characterize trace elements
• Pharmacokinetics
6© 2017 Editas Medicine
Presentation Overview
Sickle Cell Disease, b-globin Gene Regulation, and Editing Strategy
RNP based Lead Discovery platform
Fully synthetic modular gRNA
Ex-vivo T-cell and HSC editing with Cpf1
Platform for CRISPR medicines
7© 2017 Editas Medicine
RNP lead discovery
▪ Wealth of standardized data across cell types, nucleases, gRNA formats
T1 T3 T4 T5T2
Many factors influence gRNA efficacy: gRNA sequence, Nuclease type, cell type
8© 2017 Editas MedicineCONFIDENTIAL
T-cells skew to deletions as compared to insertions in HEK-293T cellsCell type dependence
BCL11Ae−CUNAM−FUGY BCL11Ae−FUVOJ−LEDA BCL11Ae−GEKOD−NYDO BCL11Ae−GUDUN−XORA
BCL11Ae−HUKAT−PAPA BCL11Ae−HUSYB−SALA BCL11Ae−HYFIH−VOMI BCL11Ae−KAQYV−GEZY
BCL11Ae−LUQUG−CISY BCL11Ae−MUXID−TYGE BCL11Ae−NAQOW−VUNA BCL11Ae−PIDAR−GIXU
BCL11Ae−QABOV−KOZA BCL11Ae−QUTYJ−GIRA BCL11Ae−RETEQ−CEFU BCL11Ae−VAJOT−JEGI
BCL11Ae−XOVIS−GUHA BCL11Ae−ZUJUP−LEBA
0.00
0.01
0.02
0.03
0.04
0.00
0.01
0.02
0.03
0.00
0.01
0.02
0.03
0.00
0.01
0.02
0.03
0.00
0.01
0.02
0.00
0.01
0.02
0.03
0.00
0.01
0.02
0.03
0.04
0.00
0.01
0.02
0.03
0.04
0.00
0.01
0.02
0.03
0.00
0.01
0.02
0.00
0.01
0.02
0.03
0.00
0.01
0.02
0.03
0.000
0.005
0.010
0.015
0.020
0.025
0.00
0.01
0.02
0.03
0.00
0.01
0.02
0.03
0.04
0.00
0.01
0.02
0.03
0.00
0.01
0.02
0.00
0.01
0.02
0.03
−60 −40 −20 0 20 40 −150 −100 −50 0 50 −100 −50 0 50 −25 0 25 50
−200 −150 −100 −50 0 50 −100 −50 0 50 −50 −25 0 25 50 −25 0 25 50
−50 −25 0 25 50 −50 −25 0 25 50 −25 0 25 50 −25 0 25 50
−200 −150 −100 −50 0 50 −30 0 30 60 −25 0 25 50 −200 −150 −100 −50 0 50
−150 −100 −50 0 50 −250 −200 −150 −100 −50 0 50Size
dens
ity
Cell.TypeHEK293T cell
Edit change in size (bp)
InsertionsDeletions
Prob
abilit
y de
nsity
Cell type
Guide 1 Guide 2 Guide 3
Guide 4 Guide 5 Guide 6
9© 2017 Editas Medicine
Normalized indel distributions display range of levels of control
Guide dependence of edit
More control Less control
Normalized edit profiles
10© 2017 Editas Medicine© CONFIDENTIAL 2016 Editas Medicine
▪ Spy-Cas9 shows the +1 insertion more often than Spy-Cas9
Indel pattern dependence on nuclease choice
10
%age of indelswith a specific edit
+1 insertions
11© 2017 Editas Medicine
Pyogenes cuts with a 1bp stagger more often than Sau Cas9
+1 overhangs a result of nuclease properties
In vivo genome editing using Staphylococcus aureus Cas9.Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F.Nature. 2015 Apr 9;520(7546):186-91. doi: 10.1038/nature14299.
+1 insertions come from base 5’ of cut site
12© 2017 Editas Medicine
Presentation Overview
Sickle Cell Disease, b-globin Gene Regulation, and Editing Strategy
RNP based Lead Discovery platform
Fully synthetic modular gRNA
Ex-vivo T-cell and HSC editing with Cpf1
Platform for CRISPR medicines
13© 2017 Editas Medicine
Maturation of the synthetic gRNA field allow for multiple formats
▪ Chemical synthesis of gRNA allows for greater flexibility, fidelity , scale and purity
▪ Synthetic chemistry goes from 3’ to 5’ , 100-mers were challenging till recently
▪ Safety and Specificity of a medicine rely on a precise characterization and control of every component
gRNA component at the core of the RNP medicine
Guide Sequence
100-mer sgRNA
14© 2017 Editas Medicine
5’ 3’
5’
3’
5’
3’
+
Why make a synthetic guide?
• Targeted chemistries anywhere in the molecule
• Unhindered ends and modifications
• Scale up and purity are more compatible with CMC requirements
Generating Synthetic Covalently-Coupled Dual gRNA
A completely non-enzymatic process for guide production
covalently-coupled dual gRNA (dgRNA)
15© 2017 Editas Medicine
Cellular Editing ActivityIn vitro transcribed and synthetic covalently-coupled dgRNA are equivalent in cells
-11 -10 -9 -8 -7 -60
20
40
60
80
Log concentation RNP (M)
% E
ditin
g (T
7E1)
IVT purified by vendorIVT purified by collaborator
unligated 2 part syntheticligated 2 part synthetic
IVT purified by vendor
covalently-coupled dgRNAIVT purified by collaborator
2-part synthetic
16© 2017 Editas Medicine
gRNA purity and sequence fidelity
A
B
Covalently-Coupled dgRNA
Covalently-coupled dgRNA result in greater sequence fidelity in target region
0.0001
0.0010
0.0100
0.1000
1.0000
10.0000
100.0000
−3 −2 −1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Position
Perc
enta
geBase
ACGT+−
0.0001
0.0010
0.0100
0.1000
1.0000
10.0000
100.0000
−3 −2 −1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Position
Perc
enta
ge
BaseACGT+−
0.0001
0.0010
0.0100
0.1000
1.0000
10.0000
100.0000
−3 −2 −1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Position
Perc
enta
ge
BaseACGT+−
17© 2017 Editas Medicine
gRNA Purity and Sequence Fidelity
A
B
Covalently-Coupled dgRNA
Covalently-coupled dgRNA result in fewer truncated molecules
18© 2017 Editas Medicine
Presentation Overview
Sickle Cell Disease, b-globin Gene Regulation, and Editing Strategy
RNP based Lead Discovery platform
Fully synthetic modular gRNA
Ex-vivo T-cell and HSC editing with Cpf1
Platform for CRISPR medicines
19© 2017 Editas Medicine
▪ Smaller gRNA just 44 nucleotides▪ The business end of gRNA is at 3’▪ dsDNA Cutting profile different from Spy and Sau Cas9▪ More targets with enzyme and evolved variants
Type VI nuclease Cpf1 with many benefits
Cpf1
- WT TTTV, TTN PAMs plusTYCV/CCCC, TATV engineeredPAM variants
- Single guide with 20-24 ntprotospacer and 19-20 nt direct repeat (~40 nt)
- 5’ staggered DNA cut with 4-5 ntoverhangs
Cas9
- WT NGG PAM plus NGAN, NGCG engineered PAM variants
- Separate crRNA and tracrRNA that can be linked into one molecule (~100 nt)
- Blunt DNA cut
20© 2017 Editas Medicine
AsCpf1 emerging as the “go to” Cpf1 with Robust activity
Screening of multiple Cpf-1 orthologs and variants
0
10
20
30
40
50
60
70
80
MS1 MS5 MS11 MS18
% in
dels
by
T7E
1% indels at four matched sites in U2OS
AsCpf1 FnCpf1 LbCpf1 Lb2Cpf1 SpCas9
21© 2017 Editas Medicine
Cpf1 editing at T-cell targets
0
20
40
60
80
100
% in
dels
by
T7E
1
% indels at a targeted locus
22© 2017 Editas Medicine
Sample editing by AsCPf1 in HSCs
Ex-vivo editing of Cpf1 at sample locus
0
20
40
60
80
100
MS1 MS5 MS11 MS18
% in
dels
by
NG
S
% indels at four matched sites
AsCpf1 SpCas9
23© 2017 Editas Medicine
Summary and Conclusions
▪ A flexible platform allows for lead discovery screening and optimization at scale
▪ Wealth of standardized data allows for insights into nuclease function and its interplay with biology to engineer a therapeutic outcome
▪ Modular gRNA allow for controlling the core of the RNP medicine
▪ Cpf1 editing points to greater flexibility in developing CRISPR medicines
Poster : 7, 33 and 68