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