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1
Genome Editing: Considerations for Therapeutic Applications
Cecilia FernandezTIDES, 2017
www.editasmedicine.com
2
The Potential to Repair Any Broken Gene
Transformative new category of medicines for genetically-defined and genetically-treatable diseases
3
Editing machinery can be engineered to target nearly any genomic location
CRISPR Unlocks Genome Editing
Targeting Sequence
Guide RNA
DNA
PAM
CutSites
Nuclease
HiFi
§ Complex of nuclease and guide RNA precisely locates and cuts genomic sites§ Ability to target several sites simultaneously using multiple guide RNAs§ Nuclease can be engineered to reach more sites and to modulate cutting
PAM: Protospacer Adjacent Motif; HiFi: High Fidelity; eS: enhanced Specificity
eS
4
CorrectDisrupt Delete
Unparalleled Platform for Gene Editing Medicines
Lipid Nanoparticle ElectroporationViral Vector
Broad Range of
Sites
Wide Delivery Options
DiverseSpectrum
of Edits
~10x
SaCas9SpCas9 Editas Platform
Cpf1 Variants
SaCas9 Variants
Cpf1SpCas9 Variants
5
Lead Finding & Specificity to Select gRNA
Testing of On-Target
Cutting
Unbiased Detection of
Off-Target Cuts(e.g., GUIDE-Seq)
in silicoPrediction of Cutting Sites
Targeted Panels for Detection of
Sites from Biased & Unbiased
Screens
In silico Selection
Identify, Measure, Minimize
6
Identification of Robust gRNAsRNP (S. pyogenes) delivery to primary T cells
• Several gRNAs performed well when assessing target expression by FACS
• Sequencing of gDNAconfirmed the presence of indels
• These gRNAs were analyzed by GUIDE-seq to identify off targetsU n
trea te dP D4 9P D5 1P D5 2P D5 3P D5 5P D5 6P D9 7P D9 8P D9 9
P D1 00
P D1 01
P D1 02
P D1 03
P D1 04
0
2 0
4 0
6 0
8 0
1 0 0
gR N A ID
% PD1 Neg
ative
% N
egat
ive
No
Gui
de
gRNAs
7
Control and Specificity to Drive PrecisionGuide-seq allows identification of highly selective candidates
GUIDE-Seq Read Count
0
500
1000
A B C DGuide RNA
Rea
ds
On-Target Off-Target 1 Off-Target 2 Off-Target 3
8
How Do You Best Measure Editing?
§ Sequence anchored detection approaches are limited to:– What is between the primers and– Amplicon size
§ One cannot detect several events and has to build and “reassemble” answers from disparate technologies (e.g. ddPCR + targeted sequencing):– Large insertions – Large deletions– Inversions– Translocations
§ Wanted a size insensitive, multiplex compatible, comparatively easy single tube method that can detect all of the above events
A simple question with a complex answer
9
Uni-Directional Targeted Sequencing (UDiTaS™)
Blending tagmentation and AMP-seq
Picelli, S., et. al (2014). Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Research, 24(12), 2033–2040.
Custom Transposon
ß UMI Barcodeß Pooling Barcodeß P5
5’
5’3’
3’ ddC
Custom Transposon Genomic DNA+
Tagmentation (~ 2Kb)
PCR Round 1 (hot start)
P5 primer
Sequence specific primer
PCR Round 2
UMI P7
P7BCBCBCP7
Sequencing
10
Comparison of Small Indel MeasurementsUDiTaS correlates well with targeted sequencing and T7E1 assays
None0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8
% Indels
guide RNA #
AMP-seqT7E1UDiTaS
11
UDiTaS™ (Uni-Directional Targeted Sequencing)
A simple, robust method for capturing complex editing events in a single reaction
1. Quantitation of editing
2. Quantitation and discovery of large deletions and inversions
3. Translocation discovery and quantitation
4. Multiplexing
12
Ex Vivo Drug Development§ Engineered autologous therapy requires multiple components
EngineeredPatient
Cell
++
PatientCell
Ribonucleoprotein Particle (RNP)RNA Guided
Endonuclease
RNA Guide
High Quality Ribonucleoprotein Particle Delivery
13
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)
14
Cellular Editing Activity
In 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
15
1
5
Assessing gRNA purity and sequence fidelity
synthetic 100mer “A”
covalently-coupled dgRNA
synthetic 100mer “B”
Development of an RNA-Seq based method for gRNA QC
16
gRNA purity and sequence fidelity
A
B
Covalently-Coupled dgRNA
Covalently-coupled dgRNA result in greater sequence fidelity in target region
17
gRNA Purity and Sequence Fidelity
A
B
Covalently-Coupled dgRNA
Covalently-coupled dgRNA result in fewer truncated molecules
5e−04
0.1502
0.0432
2e−04
0.5124
1.0384
7e−04
6e−04
1.2414
0.8864
7e−04
0.0011
0.0011
3e−04
0.0016
0.0055
1.0239
0.0192
0.0028
0.0033
3e−04
5e−04
1e−04
7e−04
7e−04
0.001
0.0015
0.0056
6e−04
0.0015
1e−04
0.011
0.133
0.0244
0.0066
1e−04
0.0072
0.0012
0.0298
1.0806
0.0914
1e−04
0.0055
5e−04
0.0102
2e−04
5e−04
0.003
0.016
0.0014
2e−04
0.0045
0.001
0.0123
0.0975
0.0532
1e−04
0.0172
0.0034
0.0014
0.3896
0.0961
0.0041
0.0528
1e−04
6e−04
3e−04
0.0038
0.3176
0.0413
0.0014
1e−04
1e−04
7e−04
6e−04
0.002
0.2954
0.0215
0.0015
0.0034
1e−04
9e−04
0.005
8e−04
0.0738
0.1507
0.0297
0.0056
0.0011
0.2056
6e−04
0.1205
0.0059
2e−04
2e−04
0.0042
0.001
9e−04
1e−04
0.0473
0.0139
7e−04
1e−04
0.0011
0.0749
0.0777
2e−04
2e−04
1e−04
0.002
0.1023
0.083
1e−04
0.0018
1e−04
0.001
0.001
0.1596
0.1036
1e−04
0.0067
5e−04
0.0293
6e−04
0.0015
0.0061
1e−04
2e−04
3e−04
0.0211
0.019
0.1122
0.0418
0.0129
1e−04
0.1447
3e−04
0.0853
0.1403
1e−04
6e−04
1e−04
0.0015
2e−04
0.004
0.0044
0.2824
0.048
0.0102
0.0107
3e−04
3e−04
0.0034
0.0027
0.002
0.3779
0.6421
0.0495
0.2605
0.0011
0.0079
0.001
2e−04
1e−04
3e−04
0.0015
0.0018
0.0015
0.0096
0.001
3e−04
0.0038
2e−04
0.0029
0.0018
0.002
9e−04
0.0165
1e−04
0.0016
1e−04
0.0037
0.0053
0.2478
0.1275
0.0076
0.089
6e−04
2e−04
3e−04
7e−04
0.0045
0.243
0.029
0.0025
5e−04
1e−04
0.0037
0.0029
0.0049
0.0272
0.213
0.1773
1e−04
5e−04
1e−04
6e−04
1e−04
0.0037
0.0023
0.3073
0.073
0.0038
0.0087
2e−04
1e−04
0.0016
0.001
0.0742
0.0772
0.016
0.0849
0.2282
0.0221
0.0382
5e−04
0.0058
7e−04
0.0037
2e−04
9e−04
2e−04
2e−04
2e−04
0.1642
0.0824
0.0056
9e−04
5e−04
0.0028
2e−04
0.0105
0.0279
0.0338
0.0405
5e−04
2e−04
0.0016
0.0016
0.0398
0.0813
0.0072
2e−04
2e−04
7e−04
2e−04
0.0289
0.0014
0.003
0.2042
0.1472
0.0049
0.0012
2e−04
0.0051
0.0028
0.1094
0.1565
0.0021
0.0037
0.0026
0.0058
0.004
0.0051
5e−04
5e−04
0.0047
0.0116
0.0326
0.115
0.0091
0.024
0.0014
9e−04
7e−04
0.0019
0.007
0.0088
0.1479
0.1092
0.0179
0.0289
2e−04
5e−04
0.0065
0.0403
0.0696
0.1195
9e−04
2e−04
2e−04
0.0035
0.0126
0.6543
0.3407
0.0021
0.0361
0.0016
5e−04
5e−04
0.0044
0.004
0.0028
0.0813
0.0263
2e−04
0.0366
0.0079
0.0226
0.0587
0.099
2e−04
0.0156
0.0082
0.0161
0.007
0.0917
0.1327
5e−04
0.1281
5e−04
0.0026
5e−04
7e−04
0.0077
0.3644
0.1726
0.024
0.0424
0.0014
2e−04
7e−04
0.0116
0.1693
0.2804
0.0238
2e−04
7e−04
0.0019
0.0061
0.0012
0.0119
0.027
0.0019
0.0012
0.0033
9e−04
0.0026
0.0082
0.2387
0.1481
0.0112
0.2189
0.0014
0.0021
0.0061
0.0256
0.0321
0.0955
0.2361
0.0354
5e−04
0.0044
0.0016
0.2352
0.4466
0.0023
0.0016
7e−04
0.0033
0.0144
0.013
0.1982
0.6248
0.0414
0.1614
0.0028
2e−04
0.0026
0.024
0.0035
0.0014
7e−04
5e−04
0.0061
0.0056
7e−04
0.0109
0.0012
7e−04
0.0142
2e−04
0.0093
0.0063
0.0012
0.0263
2e−04
2e−04
9e−04
0.0077
0.0175
0.0608
0.2385
0.0196
0.1896
0.0016
2e−04
0.003
0.003
0.0033
0.2056
0.0685
0.0154
0.0075
2e−04
0.0037
0.0144
0.0424
0.0652
0.3991
0.0123
0.0026
0.0019
5e−04
0.0151
0.0033
0.2603
0.1325
0.0221
0.034
5e−04
2e−04
0.0047
0.0016
0.0349
0.1742
0.0366
0.064
0.0525
9e−04
1e−04
1e−04
1e−04
0.0982
0.0655
0.3007
0.0727
2e−04
1e−04
2e−04
0.0121
0.0086
3e−04
3e−04
1e−04
5e−04
3e−04
2e−04
0.0044
3e−04
1e−04
1e−04
1e−04
0.0049
0.0114
8e−04
1e−04
5e−04
4e−04
0.0019
3e−04
0.0058
0.0032
0.0015
1e−04
1e−04
0.0076
0.0117
0.0084
1e−04
0.0013
0.0029
1e−04
0.0054
0.0045
1e−04
4e−04
1e−04
0.0089
0.0074
0.0025
0.001
0.0148
0.0511
0.0036
1e−04
3e−04
0.0065
2e−04
0.0099
0.0082
5e−04
0.0054
0.0378
3e−04
0.0183
0.0192
3e−04
1e−04
0.0014
0.0108
1e−04
6e−04
0.0043
0.0058
0.0019
1e−04
1e−04
0.005
0.0108
0.0331
6e−04
0.001
5e−04
0.0092
0.0042
0.0079
1e−04
2e−04
0.0509
0.2056
0.0023
0.0453
5e−04
1e−04
5e−04
1e−04
1e−04
0.001
1e−04
6e−04
6e−04
1e−04
1e−04
1e−04
0.0044
0.0397
1e−04
0.0186
1e−04
0.0025
0.0034
1e−04
0.0436
0.0034
0.0172
6e−04
0.0021
0.0051
0.0022
1e−04
1e−04
0.0039
1e−04
0.0034
0.0015
0.0122
0.031
−5−4−3−2−1
12345
−5−4−3−2−1
12345
−5−4−3−2−1
12345
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
Leng
th c
hang
e
0.26
0.58
1
% error
5e−04
0.1502
0.0432
2e−04
0.5124
1.0384
7e−04
6e−04
1.2414
0.8864
7e−04
0.0011
0.0011
3e−04
0.0016
0.0055
1.0239
0.0192
0.0028
0.0033
3e−04
5e−04
1e−04
7e−04
7e−04
0.001
0.0015
0.0056
6e−04
0.0015
1e−04
0.011
0.133
0.0244
0.0066
1e−04
0.0072
0.0012
0.0298
1.0806
0.0914
1e−04
0.0055
5e−04
0.0102
2e−04
5e−04
0.003
0.016
0.0014
2e−04
0.0045
0.001
0.0123
0.0975
0.0532
1e−04
0.0172
0.0034
0.0014
0.3896
0.0961
0.0041
0.0528
1e−04
6e−04
3e−04
0.0038
0.3176
0.0413
0.0014
1e−04
1e−04
7e−04
6e−04
0.002
0.2954
0.0215
0.0015
0.0034
1e−04
9e−04
0.005
8e−04
0.0738
0.1507
0.0297
0.0056
0.0011
0.2056
6e−04
0.1205
0.0059
2e−04
2e−04
0.0042
0.001
9e−04
1e−04
0.0473
0.0139
7e−04
1e−04
0.0011
0.0749
0.0777
2e−04
2e−04
1e−04
0.002
0.1023
0.083
1e−04
0.0018
1e−04
0.001
0.001
0.1596
0.1036
1e−04
0.0067
5e−04
0.0293
6e−04
0.0015
0.0061
1e−04
2e−04
3e−04
0.0211
0.019
0.1122
0.0418
0.0129
1e−04
0.1447
3e−04
0.0853
0.1403
1e−04
6e−04
1e−04
0.0015
2e−04
0.004
0.0044
0.2824
0.048
0.0102
0.0107
3e−04
3e−04
0.0034
0.0027
0.002
0.3779
0.6421
0.0495
0.2605
0.0011
0.0079
0.001
2e−04
1e−04
3e−04
0.0015
0.0018
0.0015
0.0096
0.001
3e−04
0.0038
2e−04
0.0029
0.0018
0.002
9e−04
0.0165
1e−04
0.0016
1e−04
0.0037
0.0053
0.2478
0.1275
0.0076
0.089
6e−04
2e−04
3e−04
7e−04
0.0045
0.243
0.029
0.0025
5e−04
1e−04
0.0037
0.0029
0.0049
0.0272
0.213
0.1773
1e−04
5e−04
1e−04
6e−04
1e−04
0.0037
0.0023
0.3073
0.073
0.0038
0.0087
2e−04
1e−04
0.0016
0.001
0.0742
0.0772
0.016
0.0849
0.2282
0.0221
0.0382
5e−04
0.0058
7e−04
0.0037
2e−04
9e−04
2e−04
2e−04
2e−04
0.1642
0.0824
0.0056
9e−04
5e−04
0.0028
2e−04
0.0105
0.0279
0.0338
0.0405
5e−04
2e−04
0.0016
0.0016
0.0398
0.0813
0.0072
2e−04
2e−04
7e−04
2e−04
0.0289
0.0014
0.003
0.2042
0.1472
0.0049
0.0012
2e−04
0.0051
0.0028
0.1094
0.1565
0.0021
0.0037
0.0026
0.0058
0.004
0.0051
5e−04
5e−04
0.0047
0.0116
0.0326
0.115
0.0091
0.024
0.0014
9e−04
7e−04
0.0019
0.007
0.0088
0.1479
0.1092
0.0179
0.0289
2e−04
5e−04
0.0065
0.0403
0.0696
0.1195
9e−04
2e−04
2e−04
0.0035
0.0126
0.6543
0.3407
0.0021
0.0361
0.0016
5e−04
5e−04
0.0044
0.004
0.0028
0.0813
0.0263
2e−04
0.0366
0.0079
0.0226
0.0587
0.099
2e−04
0.0156
0.0082
0.0161
0.007
0.0917
0.1327
5e−04
0.1281
5e−04
0.0026
5e−04
7e−04
0.0077
0.3644
0.1726
0.024
0.0424
0.0014
2e−04
7e−04
0.0116
0.1693
0.2804
0.0238
2e−04
7e−04
0.0019
0.0061
0.0012
0.0119
0.027
0.0019
0.0012
0.0033
9e−04
0.0026
0.0082
0.2387
0.1481
0.0112
0.2189
0.0014
0.0021
0.0061
0.0256
0.0321
0.0955
0.2361
0.0354
5e−04
0.0044
0.0016
0.2352
0.4466
0.0023
0.0016
7e−04
0.0033
0.0144
0.013
0.1982
0.6248
0.0414
0.1614
0.0028
2e−04
0.0026
0.024
0.0035
0.0014
7e−04
5e−04
0.0061
0.0056
7e−04
0.0109
0.0012
7e−04
0.0142
2e−04
0.0093
0.0063
0.0012
0.0263
2e−04
2e−04
9e−04
0.0077
0.0175
0.0608
0.2385
0.0196
0.1896
0.0016
2e−04
0.003
0.003
0.0033
0.2056
0.0685
0.0154
0.0075
2e−04
0.0037
0.0144
0.0424
0.0652
0.3991
0.0123
0.0026
0.0019
5e−04
0.0151
0.0033
0.2603
0.1325
0.0221
0.034
5e−04
2e−04
0.0047
0.0016
0.0349
0.1742
0.0366
0.064
0.0525
9e−04
1e−04
1e−04
1e−04
0.0982
0.0655
0.3007
0.0727
2e−04
1e−04
2e−04
0.0121
0.0086
3e−04
3e−04
1e−04
5e−04
3e−04
2e−04
0.0044
3e−04
1e−04
1e−04
1e−04
0.0049
0.0114
8e−04
1e−04
5e−04
4e−04
0.0019
3e−04
0.0058
0.0032
0.0015
1e−04
1e−04
0.0076
0.0117
0.0084
1e−04
0.0013
0.0029
1e−04
0.0054
0.0045
1e−04
4e−04
1e−04
0.0089
0.0074
0.0025
0.001
0.0148
0.0511
0.0036
1e−04
3e−04
0.0065
2e−04
0.0099
0.0082
5e−04
0.0054
0.0378
3e−04
0.0183
0.0192
3e−04
1e−04
0.0014
0.0108
1e−04
6e−04
0.0043
0.0058
0.0019
1e−04
1e−04
0.005
0.0108
0.0331
6e−04
0.001
5e−04
0.0092
0.0042
0.0079
1e−04
2e−04
0.0509
0.2056
0.0023
0.0453
5e−04
1e−04
5e−04
1e−04
1e−04
0.001
1e−04
6e−04
6e−04
1e−04
1e−04
1e−04
0.0044
0.0397
1e−04
0.0186
1e−04
0.0025
0.0034
1e−04
0.0436
0.0034
0.0172
6e−04
0.0021
0.0051
0.0022
1e−04
1e−04
0.0039
1e−04
0.0034
0.0015
0.0122
0.031
−5−4−3−2−1
12345
−5−4−3−2−1
12345
−5−4−3−2−1
12345
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
Leng
th c
hang
e
0.26
0.58
1
% error
5e−04
0.1502
0.0432
2e−04
0.5124
1.0384
7e−04
6e−04
1.2414
0.8864
7e−04
0.0011
0.0011
3e−04
0.0016
0.0055
1.0239
0.0192
0.0028
0.0033
3e−04
5e−04
1e−04
7e−04
7e−04
0.001
0.0015
0.0056
6e−04
0.0015
1e−04
0.011
0.133
0.0244
0.0066
1e−04
0.0072
0.0012
0.0298
1.0806
0.0914
1e−04
0.0055
5e−04
0.0102
2e−04
5e−04
0.003
0.016
0.0014
2e−04
0.0045
0.001
0.0123
0.0975
0.0532
1e−04
0.0172
0.0034
0.0014
0.3896
0.0961
0.0041
0.0528
1e−04
6e−04
3e−04
0.0038
0.3176
0.0413
0.0014
1e−04
1e−04
7e−04
6e−04
0.002
0.2954
0.0215
0.0015
0.0034
1e−04
9e−04
0.005
8e−04
0.0738
0.1507
0.0297
0.0056
0.0011
0.2056
6e−04
0.1205
0.0059
2e−04
2e−04
0.0042
0.001
9e−04
1e−04
0.0473
0.0139
7e−04
1e−04
0.0011
0.0749
0.0777
2e−04
2e−04
1e−04
0.002
0.1023
0.083
1e−04
0.0018
1e−04
0.001
0.001
0.1596
0.1036
1e−04
0.0067
5e−04
0.0293
6e−04
0.0015
0.0061
1e−04
2e−04
3e−04
0.0211
0.019
0.1122
0.0418
0.0129
1e−04
0.1447
3e−04
0.0853
0.1403
1e−04
6e−04
1e−04
0.0015
2e−04
0.004
0.0044
0.2824
0.048
0.0102
0.0107
3e−04
3e−04
0.0034
0.0027
0.002
0.3779
0.6421
0.0495
0.2605
0.0011
0.0079
0.001
2e−04
1e−04
3e−04
0.0015
0.0018
0.0015
0.0096
0.001
3e−04
0.0038
2e−04
0.0029
0.0018
0.002
9e−04
0.0165
1e−04
0.0016
1e−04
0.0037
0.0053
0.2478
0.1275
0.0076
0.089
6e−04
2e−04
3e−04
7e−04
0.0045
0.243
0.029
0.0025
5e−04
1e−04
0.0037
0.0029
0.0049
0.0272
0.213
0.1773
1e−04
5e−04
1e−04
6e−04
1e−04
0.0037
0.0023
0.3073
0.073
0.0038
0.0087
2e−04
1e−04
0.0016
0.001
0.0742
0.0772
0.016
0.0849
0.2282
0.0221
0.0382
5e−04
0.0058
7e−04
0.0037
2e−04
9e−04
2e−04
2e−04
2e−04
0.1642
0.0824
0.0056
9e−04
5e−04
0.0028
2e−04
0.0105
0.0279
0.0338
0.0405
5e−04
2e−04
0.0016
0.0016
0.0398
0.0813
0.0072
2e−04
2e−04
7e−04
2e−04
0.0289
0.0014
0.003
0.2042
0.1472
0.0049
0.0012
2e−04
0.0051
0.0028
0.1094
0.1565
0.0021
0.0037
0.0026
0.0058
0.004
0.0051
5e−04
5e−04
0.0047
0.0116
0.0326
0.115
0.0091
0.024
0.0014
9e−04
7e−04
0.0019
0.007
0.0088
0.1479
0.1092
0.0179
0.0289
2e−04
5e−04
0.0065
0.0403
0.0696
0.1195
9e−04
2e−04
2e−04
0.0035
0.0126
0.6543
0.3407
0.0021
0.0361
0.0016
5e−04
5e−04
0.0044
0.004
0.0028
0.0813
0.0263
2e−04
0.0366
0.0079
0.0226
0.0587
0.099
2e−04
0.0156
0.0082
0.0161
0.007
0.0917
0.1327
5e−04
0.1281
5e−04
0.0026
5e−04
7e−04
0.0077
0.3644
0.1726
0.024
0.0424
0.0014
2e−04
7e−04
0.0116
0.1693
0.2804
0.0238
2e−04
7e−04
0.0019
0.0061
0.0012
0.0119
0.027
0.0019
0.0012
0.0033
9e−04
0.0026
0.0082
0.2387
0.1481
0.0112
0.2189
0.0014
0.0021
0.0061
0.0256
0.0321
0.0955
0.2361
0.0354
5e−04
0.0044
0.0016
0.2352
0.4466
0.0023
0.0016
7e−04
0.0033
0.0144
0.013
0.1982
0.6248
0.0414
0.1614
0.0028
2e−04
0.0026
0.024
0.0035
0.0014
7e−04
5e−04
0.0061
0.0056
7e−04
0.0109
0.0012
7e−04
0.0142
2e−04
0.0093
0.0063
0.0012
0.0263
2e−04
2e−04
9e−04
0.0077
0.0175
0.0608
0.2385
0.0196
0.1896
0.0016
2e−04
0.003
0.003
0.0033
0.2056
0.0685
0.0154
0.0075
2e−04
0.0037
0.0144
0.0424
0.0652
0.3991
0.0123
0.0026
0.0019
5e−04
0.0151
0.0033
0.2603
0.1325
0.0221
0.034
5e−04
2e−04
0.0047
0.0016
0.0349
0.1742
0.0366
0.064
0.0525
9e−04
1e−04
1e−04
1e−04
0.0982
0.0655
0.3007
0.0727
2e−04
1e−04
2e−04
0.0121
0.0086
3e−04
3e−04
1e−04
5e−04
3e−04
2e−04
0.0044
3e−04
1e−04
1e−04
1e−04
0.0049
0.0114
8e−04
1e−04
5e−04
4e−04
0.0019
3e−04
0.0058
0.0032
0.0015
1e−04
1e−04
0.0076
0.0117
0.0084
1e−04
0.0013
0.0029
1e−04
0.0054
0.0045
1e−04
4e−04
1e−04
0.0089
0.0074
0.0025
0.001
0.0148
0.0511
0.0036
1e−04
3e−04
0.0065
2e−04
0.0099
0.0082
5e−04
0.0054
0.0378
3e−04
0.0183
0.0192
3e−04
1e−04
0.0014
0.0108
1e−04
6e−04
0.0043
0.0058
0.0019
1e−04
1e−04
0.005
0.0108
0.0331
6e−04
0.001
5e−04
0.0092
0.0042
0.0079
1e−04
2e−04
0.0509
0.2056
0.0023
0.0453
5e−04
1e−04
5e−04
1e−04
1e−04
0.001
1e−04
6e−04
6e−04
1e−04
1e−04
1e−04
0.0044
0.0397
1e−04
0.0186
1e−04
0.0025
0.0034
1e−04
0.0436
0.0034
0.0172
6e−04
0.0021
0.0051
0.0022
1e−04
1e−04
0.0039
1e−04
0.0034
0.0015
0.0122
0.031
−5−4−3−2−1
12345
−5−4−3−2−1
12345
−5−4−3−2−1
12345
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
Leng
th c
hang
e
0.26
0.58
1
% error
18
32523.0079
32707.085032780.6450
-‐MS, 1.4-‐1.6min, Deconvoluted (MaxEnt, 606.93-‐2003.56, *0.375, 10000)
0.0
0.5
1.0
1.5
2.0
2.5
5x10Intens.
31000 31250 31500 31750 32000 32250 32500 32750 33000 33250 m/z
Orthogonal Analytics Demonstrates Superior Material
wgs022-A_1-b,1_01_6983.d: TIC -All MS
0
1
2
3
4
7x10Intens.
1 2 3 4 5 Time [min]
wgs022-C_1-b,3_01_6991.d: TIC -All MS
0
2
4
6
7x10Intens.
1 2 3 4 5 Time [min]
wgs022-B_1-b,2_01_6987.d: TIC -All MS
0
2
4
6
7x10Intens.
1 2 3 4 5 Time [min]
A
B
Covalently-coupled dgRNA
32047.5554
32355.9671
32393.6721
32701.1024
-‐MS, 1.3-‐1.7min, Deconvoluted (MaxEnt, 594.35-‐2004.68, *0.375, 10000)
0
1
2
3
4x10Intens.
31800 32000 32200 32400 32600 32800 m/z
31146.7258 31270.0296 31475.981331781.8774
32127.1843
32310.3252
32455.7245
-‐MS, 1.4-‐1.5min, Deconvoluted (MaxEnt, 594.37-‐2004.74, *0.375, 10000)
0
1
2
3
4
4x10Intens.
30800 31000 31200 31400 31600 31800 32000 32200 32400 32600 m/z
Total Ion Chromatograph Deconvoluted Mass Spectrum
19
CorrectDisrupt Delete
Unparalleled Platform for Gene Editing Medicines
Lipid Nanoparticle ElectroporationViral Vector
Broad Range of
Sites
Wide Delivery Options
DiverseSpectrum
of Edits
~10x
SaCas9SpCas9 Editas Platform
Cpf1 Variants
SaCas9 Variants
Cpf1SpCas9 Variants
20
Cas9 Stimulates the Endogenous Repair Pathways
C-NHEJResection
Locus UnalteredSmall DeletionsSmall Insertions
3’
HRSSA
Large Deletions Correction
MMEJBlunt EJ SD-MMEJ
Alt-NHEJ
3’
Deletions InsertionsDeletions
HDR
DSB5’
WT Cas9
5’
21
Cas9 is a Flexible ToolWT Cas9
Blunt
5’
• Could we engage different pathways by using these different variants?
• Could we selectively stimulate HDR?
D10A Nickases
5’ Overhang
5’
5’
N863A Nickases
3’ Overhang
5’
3’
22
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
WT N863A D10A
DSBs Generated by D10A are Predominantly Repaired by HDR
InsertionsDeletions
HDR
N863A D10AWT
Mod
ifica
tion
(%)
3’ 5’Bothmer et al., Nat Comm 2017
23
Do Gene Conversion and Gene Correction have the same Genetic Requirement?
Gene Conversion
HBB HBD
Do they both dependent on the HR pathway?
Gene Correction
ssODN
24
Neg.Cont.
K.D.Rad51
FFRad51
Brca2
0
10
20
30
Gene Correction
1000 bp donor
1000 bp donor0
10
20
FFRad51
Brca2
0
10
20
30
Neg.Cont.
K.D.Rad51
Gene Conversion
FFRad51
Brca2
0
10
20
30
Neg.Cont.
K.D.Rad51
Gene Conversion
SS ODN donor Endogenous HBD Plasmid donor
Gene Conversion and Gene Correction have Different Genetic Requirements
HR is required for repair from double stranded donors (endogenous homology tracks or plasmids) but not single stranded donors
25
Conclusions from the Dual Nick Analysis
• Different ends activate different DNA repair pathways
• Different donors stimulate different pathwaysGene Correction mediated by ssODN is not HR dependent
C-NHEJ
Alt-NHEJ HDR
D10A Nickases
5’
N863A Nickases
5’
WT Cas9
5’
Bothmer et al., Nat Comm 2017
26
Summary
Hayat AbdulkerimLuis BarreraAnne BothmerFrank BuquicchioDawn CiullaCecilia Cotta-RamusinoGeorgia GiannoukosKiran Gogi
Fred HarbinskiHari JayaramEugenio MarcoCarrie MarguliesVic MyerTanushree PhadkeTerence TaChris Wilson
i2 Pharmaceuticals team
§ We are developing precise and durable therapies by selecting and directing nuclease activity, delivery and the cellular repair response.
§ We can effectively measure off-target and other genomic outcomes.§ We can make fully synthetic covalently-coupled dgRNAs of high quality.§ Our understanding of how different lesions are repaired and the connection with the nature of the donor allow us to engineer for the desired outcomes.