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.