characterization of cas9-mediated genome editing in human t … · 2020. 8. 10. ·...

1
Characterization of Cas9-mediated genome editing in human T cells G. Grant Welstead 1 , Jennifer L. Gori 1 , Justin Fang 1 , McKensie Collins 1 , Will Selleck 1 , Ari Friedland 1 , Hari Jayaram 1 , David Bumcrot 1 1 Editas Medicine, 300 3rd Street, Cambridge, MA 02142 Identification of efficient gRNAs in 293s Comparison of Cas9-mediated editing by RNP and mRNA in Jurkat T cells Generation of CD3 - cells by RNP delivery to Jurkat T cells Successful editing of naive T cells by RNP delivery Generation of CD3 - cells by mRNA/gRNA delivery to activated CD4 + T cells Loss of CD3 expression 0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00% 14.00% 16.00% 18.00% 20.00% TRBC-210 TRAC-4 AAVS1 % CD3 - cells Day2 Day3 TRAC locus analysis 0.0 0.5 1.0 1.5 2.0 2.5 TRBC-210 TRAC-4 AAVS1 %NHEJ 0.0 1.0 2.0 3.0 4.0 5.0 6.0 % NHEJ TRBC-210 TRAC-4 AAVS1 TRBC2 locus analysis 0.00E+00 2.00E+05 4.00E+05 6.00E+05 8.00E+05 1.00E+06 1.20E+06 1.40E+06 1.60E+06 Day 1 Day 2 Day 3 TRBC-210 TRAC-4 AAVS1 TRAC locus analysis 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 Nega ve Ctl TRAC-233 % NHEJ TRAC-233 0.00E+00 5.00E+06 1.00E+07 1.50E+07 2.00E+07 2.50E+07 3.00E+07 Day 1 Day 2 Day 3 Total # of cells TRAC-233 Untreated 0 5 10 15 20 25 30 35 40 45 50 % NHEJ 0 5 10 15 20 25 30 35 40 45 50 TRAC-148 TRAC-147 TRAC-234 TRAC-167 TRAC-177 TRAC-176 TRAC-257 TRAC-233 TRAC-231 TRAC-163 TRAC-241 TRAC-179 TRAC-178 % NHEJ 0 2 4 6 8 10 12 14 16 TRBC-40 TRBC-52 TRBC-25 TRBC-35 TRBC-50 TRBC-39 TRBC-49 TRBC-51 TRBC-26 TRBC-47 TRBC-45 TRBC-34 TRBC-227 TRBC-41 TRBC-30 TRBC-206 TRBC-32 TRBC-276 TRBC-274 TRBC-230 TRBC-235 TRBC-38 TRBC-223 TRBC-221 TRBC-48 TRBC-216 TRBC-210 TRBC-268 TRBC-193 TRBC-246 TRBC-228 TRBC-43 TRBC-272 TRBC-33 TRBC-44 TRBC-211 TRBC-253 TRBC-18 TRBC-6 TRBC-85 TRBC-129 TRBC-93 % NHEJ TRBC2 TRBC1 0 2 4 6 8 10 12 14 16 18 20 % NHEJ 0 5 10 15 20 25 % NHEJ 0 5 10 15 20 25 30 35 40 45 50 % NHEJ S. aureus S. pyogenes TRBC gRNAs TRAC gRNAs PD-1 gRNAs 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 Day 1 Day 2 Day 3 Untreated Jurkat %NHEJ mRNA RNP 0.00E+00 2.00E+06 4.00E+06 6.00E+06 8.00E+06 1.00E+07 1.20E+07 1.40E+07 Day 1 Day 2 Day 3 mRNA RNP mRNA RNP d e t a e r t n U 3 y a D 3 y a D 2 y a D 2 y a D 1 y a D 1 y a D Summary A A) 4x 10^6 Naïve CD3+ T cells ewere lectroporated with S. aureus Cas9/gRNA - - - locus. 293s were transfected with two plasmids – one encoding S. aureus or S. pyogenes Cas9 and the other encoding the listed gRNA. The graph sum- marizes the average %NHEJ observed at the specificied locus for each gRNA, which was cal- culated from a T7E1 assay performed on genom- ic DNA isolated from duplicate samples. A) S. aureus Cas9 and TRAC-233 gRNA were mixed and allowed to form complexes for 10 min. Prior to introduction to the cells, a small amount was removed to analyze in a DSF assay. B) Jurkat T cells electroporated with S. aureus Cas9/gRNA (TRAC-233) RNPs target- ing TRAC were cultured in RPMI1640 and counted for 3 consecutive days following stain- ing with trypan blue. C) Transduced Jurkat cells were stained with an APC-CD3 antibody and analyzed by FACS. The cells were ana- lyzed on day 1, day 2 and day 3 after the elec- troporation. D) Quantification of the CD3 nega- tive population from the plots in (C). E) % NHEJ results from the T7E1 assay performed on the TRAC locus. A) Jurkat T cells were electroporated with either S. pyogenes Cas9 mRNA and PDCD1 gRNA (P- DCD1-108) or S. pyogenes Cas9/gRNA (PD- CD1-108) RNP targeting PDCD1. Cells were re- plated in RPMI1640 and counted for 3 consecu- tive days after staining with trypan blue. B) Gel image of the DNA resulting from the T7E1 assay performed on the PDCD1 locus at 24, 48, and 72 hours. C) Quantification of %NHEJ results from the gel depicted in (B). Higher levels of %NHEJ were detected with RNP vs mRNA delivery. As ex- pected, there was undetectable levels of NHEJ in untreated Jurkat cells. 0.0 5.0 10.0 15.0 20.0 25.0 Untreated TRAC-233 %NHEJ TRAC locus analysis 0.0% 5.0% 10.0% 15.0% 20.0% 25.0% 30.0% Untreated TRAC-233 % CD3- cells Day 1 Day 2 Day 3 CD3 - expression 0.00E+00 2.00E+06 4.00E+06 6.00E+06 8.00E+06 1.00E+07 1.20E+07 1.40E+07 1.60E+07 Day 1 Day 2 Day 3 Untreated TRAC-233 s l l e C f o # l a t o T Day 1 Day 2 Day 3 Untreated TRAC-233 CD3 expression CD3 expression A B C D E A) Activated CD4+ T cells were electroporated with S. pyogenes Cas9 mRNA and the gRNA indicated (TRBC-210, TRAC-4 or AAVS1). Cells were replated in RPMI1640 + IL-2 and counted for 3 consecutive days after staining with trypan blue. B) The electroporated cells were collected at day 2 and day 3 post electroporation and were stained with an APC-CD3 antibody and analyzed by FACS. Quantification of the CD3 negative population is shown. C) %NHEJ results from the T7E1 assay performed on TRBC2 and TRAC loci. B C Cas9-mediated T cell editing using Cas9 from both S. aureus and S. pyogenes. Delivery of Cas9 as an RNP yields better viability and cutting efficiency for the PDCD1 gRNA, PDCD1-108. Nearly 45% gene editing in Jurkat T cells at the PDCD1 locus Generation of nearly 25% CD3 Jurkat T cells using RNP delivery Generation of approximately 18% CD3 - T cells when targeting the TRBC locus in activated T CD4 T cells Over 15% gene editing in naïve T cells was achieved using RNP against TRAC All electroporation experiments in T cells were performed using the Maxcyte system. The authors would like to thank members of the Maxcyte team that facili- tated our experiments - Linhong Li and Madhusudan V. Peshwa. We would also like to thank Shondra Miller at the Washington University Genome En- gineering and IPSC Center for providing Editas with some reagents used in our ex- periments. Acknowledgements Abstract Genome editing via CRISPR/Cas9 promises to provide a novel class of therapies for a variety of human diseases. To unlock the potential of the CRISPR/Cas9 technology, a deeper under- standing of its efficacy in different primary cell types is required. A cell type of particular interest for gene editing is the human T cell due to its central role in the evolving cancer immunotherapy field. To better understand the utility of Cas9-mediated gene editing for engineering human T cells, we surveyed a variety of delivery modalities including electroporation of RNA and adminis- tration of ribonucleic acid-protein complexes. Additionally, we assessed the functionality of different Cas9 variants in human T cells. Here we report our findings including genome editing in human T cells using CRISPR/Cas technology. 0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0% Day 2 Day 4 % Viability TRAC-233 Untreated D A B C A B C

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Page 1: Characterization of Cas9-mediated genome editing in human T … · 2020. 8. 10. · Characterization of Cas9-mediated genome editing in human T cells G. Grant Welstead1, Jennifer

Characterization of Cas9-mediated genome editing in human T cellsG. Grant Welstead1, Jennifer L. Gori1, Justin Fang1, McKensie Collins1, Will Selleck1, Ari Friedland1, Hari Jayaram1, David Bumcrot1

1Editas Medicine, 300 3rd Street, Cambridge, MA 02142

Identification of efficient gRNAs in 293s

Comparison of Cas9-mediated editing by RNP and mRNA in Jurkat T cells

Generation of CD3- cells by RNP delivery to Jurkat T cells

Successful editing of naive T cells by RNP delivery

Generation of CD3- cells by mRNA/gRNA delivery to activated CD4+ T cellsLoss of CD3 expression

0.00%

2.00%

4.00%

6.00%

8.00%

10.00%

12.00%

14.00%

16.00%

18.00%

20.00%

TRBC-210 TRAC-4 AAVS1

% C

D3-ce

lls

Day2

Day3 TRAC locus analysis

0.0

0.5

1.0

1.5

2.0

2.5

TRBC-210 TRAC-4 AAVS1

%N

HEJ

0.0

1.0

2.0

3.0

4.0

5.0

6.0

% N

HEJ

TRBC-210 TRAC-4 AAVS1

TRBC2 locus analysis

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

1.40E+06

1.60E+06

Day 1 Day 2 Day 3

TRBC-210 TRAC-4 AAVS1

TRAC locus analysis

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

Nega ve Ctl TRAC-233

% N

HEJ

TRAC-2330.00E+00

5.00E+06

1.00E+07

1.50E+07

2.00E+07

2.50E+07

3.00E+07

Day 1 Day 2 Day 3

Tota

l # o

f cel

ls

TRAC-233 Untreated

0

5

10

15

20

25

30

35

40

45

50

% N

HEJ

0

5

10

15

20

25

30

35

40

45

50

TRAC-148 TRAC-147 TRAC-234 TRAC-167 TRAC-177 TRAC-176 TRAC-257 TRAC-233 TRAC-231 TRAC-163 TRAC-241 TRAC-179 TRAC-178

% N

HEJ

0

2

4

6

8

10

12

14

16

TRBC

-40

TRBC

-52

TRBC

-25

TRBC

-35

TRBC

-50

TRBC

-39

TRBC

-49

TRBC

-51

TRBC

-26

TRBC

-47

TRBC

-45

TRBC

-34

TRBC

-227

TRBC

-41

TRBC

-30

TRBC

-206

TRBC

-32

TRBC

-276

TRBC

-274

TRBC

-230

TRBC

-235

TRBC

-38

TRBC

-223

TRBC

-221

TRBC

-48

TRBC

-216

TRBC

-210

TRBC

-268

TRBC

-193

TRBC

-246

TRBC

-228

TRBC

-43

TRBC

-272

TRBC

-33

TRBC

-44

TRBC

-211

TRBC

-253

TRBC

-18

TRBC

-6

TRBC

-85

TRBC

-129

TRBC

-93

% NH

EJ

TRBC2

TRBC1

0

2

4

6

8

10

12

14

16

18

20

% NH

EJ

0

5

10

15

20

25

% N

HEJ

0

5

10

15

20

25

30

35

40

45

50

% N

HEJ

S. aureus

S. pyogenes

TRBC gRNAs TRAC gRNAs PD-1 gRNAs

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

Day 1 Day 2 Day 3 Untreated Jurkat

%N

HEJ

mRNA RNP

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07

1.40E+07

Day 1 Day 2 Day 3

mRNA RNP

mRNA RNP

detaertnU

3 yaD

3 yaD

2 yaD

2 yaD

1 yaD

1 yaD

Summary

A A) 4x 10^6 Naïve CD3+ T cells ewere lectroporated with S. aureus Cas9/gRNA

-

-

-

locus.

293s were transfected with two plasmids – one

encoding S. aureus or S. pyogenes Cas9 and the

other encoding the listed gRNA. The graph sum-

marizes the average %NHEJ observed at the

specificied locus for each gRNA, which was cal-

culated from a T7E1 assay performed on genom-

ic DNA isolated from duplicate samples.

A) S. aureus Cas9 and TRAC-233 gRNA were

mixed and allowed to form complexes for 10

min. Prior to introduction to the cells, a small

amount was removed to analyze in a DSF

assay. B) Jurkat T cells electroporated with S.

aureus Cas9/gRNA (TRAC-233) RNPs target-

ing TRAC were cultured in RPMI1640 and

counted for 3 consecutive days following stain-

ing with trypan blue. C) Transduced Jurkat

cells were stained with an APC-CD3 antibody

and analyzed by FACS. The cells were ana-

lyzed on day 1, day 2 and day 3 after the elec-

troporation. D) Quantification of the CD3 nega-

tive population from the plots in (C). E) %

NHEJ results from the T7E1 assay performed

on the TRAC locus.

A) Jurkat T cells were electroporated with either

S. pyogenes Cas9 mRNA and PDCD1 gRNA (P-

DCD1-108) or S. pyogenes Cas9/gRNA (PD-

CD1-108) RNP targeting PDCD1. Cells were re-

plated in RPMI1640 and counted for 3 consecu-

tive days after staining with trypan blue. B) Gel

image of the DNA resulting from the T7E1 assay

performed on the PDCD1 locus at 24, 48, and 72

hours. C) Quantification of %NHEJ results from

the gel depicted in (B). Higher levels of %NHEJ

were detected with RNP vs mRNA delivery. As ex-

pected, there was undetectable levels of NHEJ in

untreated Jurkat cells.

0.0

5.0

10.0

15.0

20.0

25.0

Untreated TRAC-233

%N

HEJ

TRAC locus analysis

0.0%

5.0%

10.0%

15.0%

20.0%

25.0%

30.0%

Untreated TRAC-233

% C

D3-c

ells

Day 1 Day 2 Day 3

CD3- expression

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

1.20E+07

1.40E+07

1.60E+07

Day 1 Day 2 Day 3

Untreated TRAC-233

slleC fo # l at oT

Day 1 Day 2 Day 3

Untreated

TRAC-233

CD3 e

xpre

ssion

CD3 e

xpre

ssion

A

B

C

D

E

A) Activated CD4+ T cells were electroporated with S. pyogenes Cas9 mRNA and the gRNA indicated (TRBC-210, TRAC-4 or AAVS1). Cells were replated in RPMI1640 + IL-2 and counted for 3 consecutive days after staining with trypan blue. B) The electroporated

cells were collected at day 2 and day 3 post electroporation and were stained with an APC-CD3 antibody and analyzed by FACS. Quantification of the CD3 negative population is shown. C) %NHEJ results from the T7E1 assay performed on TRBC2 and TRAC loci.

B

C

Cas9-mediated T cell editing using Cas9 from both S. aureus and S. pyogenes.

Delivery of Cas9 as an RNP yields better viability and cutting efficiency for the PDCD1 gRNA, PDCD1-108.

Nearly 45% gene editing in Jurkat T cells at the PDCD1 locus

Generation of nearly 25% CD3 Jurkat T cells using RNP delivery

Generation of approximately 18% CD3- T cells when targeting the TRBC locus in activated T CD4 T cells

Over 15% gene editing in naïve T cells was achieved using RNP against TRAC

All electroporation experiments in T cells were performed using the Maxcyte system. The authors would like to thank members of the Maxcyte team that facili-tated our experiments - Linhong Li and Madhusudan V. Peshwa.

We would also like to thank Shondra Miller at the Washington University Genome En-gineering and IPSC Center for providing Editas with some reagents used in our ex-periments.

Acknowledgements

AbstractGenome editing via CRISPR/Cas9 promises to provide a novel class of therapies for a variety of human diseases. To unlock the potential of the CRISPR/Cas9 technology, a deeper under-

standing of its efficacy in different primary cell types is required. A cell type of particular interest for gene editing is the human T cell due to its central role in the evolving cancer immunotherapy

field. To better understand the utility of Cas9-mediated gene editing for engineering human T cells, we surveyed a variety of delivery modalities including electroporation of RNA and adminis-

tration of ribonucleic acid-protein complexes. Additionally, we assessed the functionality of different Cas9 variants in human T cells. Here we report our findings including genome editing in

human T cells using CRISPR/Cas technology.

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

Day 2 Day 4

% V

iabi

lity

TRAC-233 Untreated

D

A B C

A B C