TCRα - GFP CD3D - GFP CD3E - GFP CD3G - GFP CD3Z - GFP No HDRT Control HDR Template a Default CD8 T Cells TCR No Stim Complex

Size + With stim 50.2% 33.1% 50.4% 31.5% 28.3% 0.1% GFP / tNGFR (CD4) CD4 T Cells b IL2RA-N - tNGFR IL2RA-C - GFP IL2RB - tNGFR IL2RG - tNGFR CD4 - GFP CD8α - GFP CD45 - GFP B2M - tNGFR

50.1% + 29.5% 39.9% 25.4% (CD4) 34.2% 29.3% 47.5% 47.6% CD5 - tNGFR CD27 - tNGFR CD28 - tNGFR CD48 - tNGFR CCR2 - tNGFR CCR7 - tNGFR CX3CR1 - tNGFR CXCR4 - tNGFR

Surface Receptors

20.9% 2.3% 52.3% 22.8% (CD4) 8.8% 35.2% 0.04% 20.4%

ICOS - tNGFR OX40 - GFP 41BB - GFP IL7R - tNGFR ITGB1 - tNGFR ITGB7 - tNGFR LTK - tNGFR CTNNB1 - GFP

42.0% 0.5% + 8.7% 42.7% 10.5% 27.9% 0.1% + 2.9% c CTLA4-N - tNGFR CTLA4-C - GFP PD1-N - tNGFR PD1-C - GFP TIM3 - tNGFR LAG3 - tNGFR TIGIT - GFP Checkpoint Receptors

+ 8.7% + 10.5% +(CD4) 11.1% +(CD4) 3.9% + 24.3% + 38.9% + 2.2% d STAT1 - tNGFR STAT2 - tNGFR STAT4 - tNGFR STAT5B-N - GFP STAT5B-C - GFP STAT6 - tNGFR FOXO1 - tNGFR FOXP1 - tNGFR

Transcription 49.5% 12.6% 32.3% 4.8% 21.9% + 33.2% + 19.6% (CD4) 11.9% Factors JUN - GFP JUNB - tNGFR JUND - tNGFR IRF4 - tNGFR BATF - GFP TRIM22 - GFP SATB1 - GFP NCOA3 - GFP

+ 11.6% + 35.7% 1.9% + 16.3% +(CD4) 49.3% 11.7% + 48.2% +(CD4) 15.4% e ACTB - GFP TUBA1B - GFP CLTA - GFP LMNB1 - GFP PXN - GFP MAP1LC3B - GFP WAS - tNGFR Cytoskeletal Components

29.1% 39.9% 31.7% 35.6% (CD4) 17.1% + 30.8% 31.3% f HIST1H2BJ - GFP HIST2H2BE - GFP ATP2A2 - GFP HDAC3 - GFP FBL - GFP ELOB - GFP CTCF - GFP LAMP1 - GFP

43.0% 18.2% 23.0% 22.1% 24.2% 0.4% 16.1% 20.0%

KIAA1551-N - GFP KIAA1551-C - GFP TOMM20 - GFP SEC61B - GFP SLC25A17 - GFP ST6GAL1 - GFP RAB5A - GFP RAB11A - GFP

Additional + 6.7% + 3.9% 14.8% 44.2% 1.7% + 4.6% + 4.9% 53.8% Target Loci IL2 - tNGFR LCK - tNGFR CARD11 - tNGFR JAK3 - GFP CUL5 - GFP ARIH2 - GFP CBFB - GFP STRAP - GFP

+(CD4) 5.2% 35.0% 10.5% + 20.2% + 15.7% + 30.7% 15.1% 33.0% DDX20 - GFP NCOR1 - GFP TBL1XR1 - GFP NPM1 - GFP SMC1A - GFP CBX1 - GFP NUP153 - GFP FUS - GFP

+ 42.4% 6.1% 26.2% 24.4% 28.2% 20.2% + 47.9% 22.5% a b

T Cell Isolation + Activation Expansion Readouts

I I I I I I I I I I I Electroporation Resting +Stim CD4 I I I I I I I I I I I I I X X X X I I I I I I I I I I I I I I I CD8 I I I I I Day: 0 2 4 6 7 I I I I

RNA-Seq RNA-Seq RNA-Seq Flow Cytometry ~1kb Knockin 6 Donors 2 Cell Types 91 Genomic 2 gRNAs/ ATAC-Seq ATAC-Seq Amplicon-Seq (D6) Sites Site

c CD8 v CD4 T Cells 5 CD8A 4 ) 41BB

f 3 CD4 LAG3 o

/ io t 2

Ra Enriched in

(CD8 CD8 T cells in

1 + kin

c 0 no Change

K -1

% Enriched in old

F CD4 T cells -2 erved s Log2 -3 b O -4 IL2 CD4 -5

PD1-C Term d Stimulated vs Resting 5 41BB

) 4 PD1-N Term

im OX40

st f 3 o

Un io / t 2 im Ra

Enriched in St ( in Stimulated Cells 1 + 0 Change nockin -1 K

old Enriched in %

F Resting Cells -2 ed v

Log2 -3 bser

O IL7R -4 CCR2 FBL -5

e Off-Target 20.0

17.5 %

kin

c 15.0 no

t K 12.5 FBL arge T - 10.0 Off

ed 7.5 v IL2RG

bser 5.0 O STAT2

2.5

0.0 91 unique HDR templates with Scrambled gRNA f g 10000 1.00

) 0.75 M %

TP 1000 (

ing 4 tt

Cu

Day 0.50

ed v eq A S

100 bser O RN 0.25

R2 = 0.50 R2 = 0.03 10 0.00 10000 100000 1000000 0 20 40 60 80 MFI of Knockin + Cells Predicted Cut Score Parameter Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Donor 6 Donor Avg a 60 2 2 2 2 2 2 60 2 R adj = 0.0074 R adj = − 0.00081 R adj = 0.0066 R adj = 0.0082 R adj = 0.0036 R adj = 0.0073 R adj = 0.02 gRNA Cut 40 40 Distance (bps) 20 20

0 0 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 b 60 2 2 2 2 2 2 60 2 R adj = − 0.00097 R adj = − 6.2e−05 R adj = 0.0037 R adj = 0.013 R adj = 0.0012 R adj = 0.0067 R adj = − 0.0024 gRNA Cut 40 40

Direction 20 20

0 0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0 c 60 2 2 2 2 2 2 60 2 R adj = − 0.0026 R adj = − 0.0024 R adj = − 0.0032 R adj = 0.0058 R adj = − 0.0031 R adj = − 0.0029 R adj = 0.00046

gRNA 40 40 Predicted Cutting 20 20

0 0 20 30 40 50 60 70 20 30 40 50 60 70 20 30 40 50 60 70 20 30 40 50 60 70 20 30 40 50 60 70 20 30 40 50 60 70 20 30 40 50 60 70 d 60 2 2 2 2 2 2 60 2 R adj = 0.039 R adj = 0.0092 R adj = 0.034 R adj = 0.042 R adj = 0.03 R adj = 0.032 R adj = 0.059

gRNA 40 40 Observed Cutting 20 20

0 0 0.75 0.80 0.85 0.90 0.95 1.00 0.75 0.80 0.85 0.90 0.95 1.00 0.75 0.80 0.85 0.90 0.95 1.00 0.75 0.80 0.85 0.90 0.95 1.00 0.75 0.80 0.85 0.90 0.95 1.00 0.75 0.80 0.85 0.90 0.95 1.00 0.75 0.80 0.85 0.90 0.95 1.00 e 60 2 2 2 2 2 2 60 2 Target R adj = 0.14 R adj = 0.15 R adj = 0.12 R adj = 0.083 R adj = 0.17 R adj = 0.13 R adj = 0.18 Gene RNA 40 40

Expression 20 20 (TPM, Day 0) 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

60 2 2 2 2 2 2 60 2 R adj = 0.22 R adj = 0.24 R adj = 0.24 R adj = 0.21 R adj = 0.31 R adj = 0.26 R adj = 0.25

40 40 (Day 2) 20 20

0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

60 2 2 2 2 2 2 60 2 R adj = 0.25 R adj = 0.29 R adj = 0.25 R adj = 0.23 R adj = 0.34 R adj = 0.28 R adj = 0.28

40 40 (Day 4) 20 20

0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

f 60 2 2 2 2 2 2 60 2 R = 0.17 R = 0.16 R = 0.18 R = 0.23 R = 0.23 R = 0.19 R = 0.17 Target adj adj adj adj adj adj adj Gene DNA 40 40

Accessibility 20 20 (RPM, Day 0) 0 0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0

60 2 2 2 2 2 2 60 2 R adj = 0.16 R adj = 0.16 R adj = 0.19 R adj = 0.22 R adj = 0.24 R adj = 0.2 R adj = 0.18

40 40 (Day 2) 20 20

0 0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 g 60 2 2 2 2 2 2 60 2 R adj = 0.34 R adj = 0.35 R adj = 0.35 R adj = 0.34 R adj = 0.44 R adj = 0.37 R adj = 0.39 Predicted 40 40

Knockin % 20 20

0 0 Observed Knockin % 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 Parameter a 6 ELOB

JUND 4

2 Higher Predicted Knockin % Than Observed 0 Lower Predicted Knockin % (Predicted / Observed) Than Observed

Log2 Fold Change in Ratio of -2

Predicted vs Observed Knockin % ITGB1 Observed and Predicted Knockin % Averaged Across 6 Donors STAT4 at 90 unique genomic sites x 2 gRNAs per site x 2 Cell Types DDX20 -4 b c 1 1

0.5 0.5

0 0 -5 0 5 -5 -2.5 0 2.5 5 Log2(Predicted / Observed KI) Log2(Predicted / Observed KI)

CD27 DDX20

TIGIT STAT4

ST6GAL1 ITGB1

LTK STAT1

JUND PTPRC

ELOB TBL1XR1

Lower Predicted Higher Predicted Lower Predicted Higher Predicted Knockin % Knockin % Knockin % Knockin % Than Observed Than Observed Than Observed Than Observed gRNA 1 gRNA 2 gRNA 1 gRNA 2 d e

TRIM22 CD8A

IL2RB CD4

SATB1 gRNA Cut HDRT IL2

CTCF LAG3

gRNA Cut CCR7 HDRT JUN

FBL TNFRSF4

Lower Predicted Higher Predicted Lower Predicted Higher Predicted Knockin % Knockin % Knockin % Knockin % Than Observed Than Observed Than Observed Than Observed gRNA 1 gRNA 2 CD4 T Cells CD8 T Cells a GEEP with Synthetic Promoter + Endogenous Product SFFV

tNGFR 2A SFFV

Target Genomic Locus Exon 1

b SFFV Promoter PDCD1 SFFV Promoter Lag3 SFFV Promoter IL2RA Knock-in Resting Cells Unedited

PD1 Lag3 IL2RA c SFFV tNGFR PDCD1 SFFV tNGFR Lag3 SFFV tNGFR IL2RA

Control

On Target tNGFR- Resting Cells

On Target tNGFR+

Control

On Target tNGFR- Re-stimulated Cells On Target tNGFR+

PD1 Lag3 IL2RA d

Control

On Target A 3 L2R PD1 Lag I tNGFR tNGFR tNGFR e

SFFV tNGFR cJun SFFV tNGFR JunB SFFV tNGFR JunD SFFV tNGFR IRF4

12% 22.5% 15.8% 19.6% tNGFR Size a d

GEEP with Endogenous Promoter + GEEP with Synthetic Extracellular Domain + Synthetic Product at PDCD1 Gene Locus Endogenous Intracellular Domain at PDCD1 Locus

tNGFR PolyA

tNGFR 2A 2A NGFR Extracellular Domain

PDCD1 Locus

Exon 1 Exon 3

b c e

tNGFR 2A PDCD1 Exon 1 tNGFR PolyA PDCD1 Exon 1 NGFR EC PDCD1 Exon 3

0.386% 0.302% 0.521% 0.0307% 0.393% 0.0744 - Stimulation - Stimulation

1.17% 3.72% 1.3%

5.64% 13.2% 4.57% 0.843% 16.9% 4.03% + Stimulation + Stimulation

3.59% 20.3% 28.8% PD1 PD1 tNGFR tNGFR a GEEP with Endogenous Promoter + Synthetic Product

tNGFR 2A

Target Genomic Locus

Exon 1

Day 7 Day 7 CD4 CD4 + Stim CD8 CD8 + Stim b Day 3 - Stimulation + Stimulation 25k 200k 20k 150k

MFI +Stim

15k

A +Stim R R 100k

GF 10k IL2 IL2RA MFI tN 50k 5k

IL2RA 0 0 c 20k 60k

MFI 40k

R 10k GF CD28 tN CD28 MFI 20k

CD28 0 0 d 15k 60k

10k 40k MFI

R Lag3 GF Lag3 MFI 5k

tN 20k

Lag3 0 0 tNGFR 3 4 5 6 7 3 4 5 6 7 Day Post Electroporaiton Day Post Electroporaiton Control On Target a GEEP with Endogenous Specificity + Synthetic Signalling into CD3 Loci

2A BFP

CD28 2A GFP

41BB 2A mCherry

Target Genomic Locus

CD3 Final Exon

CD28-P2A-GFP 41BB-P2A-mCherry P2A-BFP b 15.3% 15.6% 19.2%

CD3 Delta

22.5% 27.4% 25.5%

CD3 Epsilon

15.5% 12.0% 12.2%

CD3 Gamma

11.4% 15.5% 11.3%

CD3 Zeta

c GFP mCherry BFP CD3 c CD3 + 41BB IC CD3 + CD28 IC

0.8 0.8 CD28 Stimulation CD3 Stimulation 0.6 0.6 CD3/CD28 Stimulation e

0.4 0.4 Chang

old

0.2 F 0.2 Log2 0.0 0.0

-0.2 -0.2

Zeta Delta Epsilon Gamma Zeta Delta Epsilon Gamma CD3 Complex Member CD3 Complex Member a

5’ Homology Arm TCRβ Insert X Insert Y TCRα VJ 3’ Homology Arm 2A 2A (tNGFR) 2A (GFP) 2A

Total Insertion Size: ~3.2 kb

Target Genomic Locus

TRAC Exon 1

b

7.8% 95.2%

83.8% 7.5% 1.4% 2.4% NY-ESO-1 GFP TCR tNGFR

0.8% 14.8%

98.6% 0.09% 78.5% 3.0% GFP GFP tNGFR tNGFR a

Unstimulated 24 hr Post Stim NY-ESO-1 tNGFR b c

100

75

50

% dnTGFR2+ (-TGFB1) 45.1 54.9 Unstimulated 25

NY-ESO-1 0 CD3 tNGFR Stimulation: - + - + TGFβ conc (ng): 0 0 25 25 a Insertion Site Genomic Locus

5’ Homology Arm 3’ Homology Arm HDR Template (Episomal) DNA Insert Unique Constant Barcode 1 HA Mismatches Not incorporated during HDR On-Target Knockin

Off-Target Knockin Constant HA Mismatches

(Homology Arms are Insert Specific Genomic Site Specific incorporated at NHEJ Forward Primer Reverse Primer mediated off-target insertions)

b c Homology Arm Unmodified 3’ HA Mismatches 1.25

22.9% 19.1% 1.00

On-Target 0.75

gRNA 0.50 0.0% 0.0%

0.25 Off-Target tNGFR

Relative HDR (vs No HA Mismatches) 0.00 NY-ESO-1 Unmodified 3’ HA Mismatches Homology Arm a b c Analysis of Pooling Stage for Pooled Knockin Screens

Condition 16.2% 42.1% 11.5% Sorted Populations

Pooled NY-ESO-1- Assembly NY-ESO-1+ RFP+ GFP- 43.5% Pooling DNA Fragments RFP- GFP+ Prior to Assembly 19.9% 42.3% 12.6%

Pooled PCR

43.7% Isolate Isolate 23.9% 42.5% 12.7% Pooling Plasmids mRNA genomic DNA Prior to PCR Pooled Electroporaiton Pooling HDRTs Prior to Electroporation 43.5% 25.1% 47.1% 0.1% cDNA Pooled Culture

Pooling Cells 50.2% Prior to Expansion Barcode PCR 23.9% 0.0% 0.0% Indexing PCR

GFP Only

98.1% Sequencing 24.8% 97.2% 0.0%

RFP Only

0.0% RFP Protocol NY-ESO-1 Step: Assembly PCR Electroporation Culture TCR GFP

e d f Sorted NY-ESO-1+ Cells 10 10 100

e gDNA ) n 8 cDNA 80 io t ode arcod c ica f B 6 ar 60 B ing mpli s or

5 A e Mis

v 4 40 i s t

(Cells Observed (Cells) Detected (Barcode- gDNA) Rela Read 2 20 Detected (Barcode- cDNA) GFP % % 0 0 0

Sorting d k d y R ro e n ly ly e - l te + C t ur In o n n rt u r t ingle- ti Condition: o -1 B o -1 P lec S k O S S E Cul P No Cells ESO ESO ssembl noc F FP O - - A K Assembly ropora G R Y Y ct N N le E Pooling Condition Pooling Condition g h Sorted GFP + Cells- gDNA Sorted RFP+ Cells- gDNA Observed Template Switching Predicted Template Switching

100 100 100 100 e e gDNA

od Donor 1 c 80 80 Donor 2 80 80 cDNA ar arcod s s

60 FP B 60 60 60 R GFP B

Read h h Read t t f f o wi wi o

40 40 40 40

% %

Reads 20 20 Reads 20 20

% % 80.4% 87.4% 95.1% 99.9% 100.0% 79.1% 87.8% 95.1% 99.7% 100.0% 0 0 0 0 y n e y y R n e y y e y R n e n Pooling R io nl R n In -I C tio tur nl C t tur C tio ur - C tio tur P P P t ck P ck Condition: Cul Cul sembl sembl Cul no ssembl ssembl FP O s Cul no A ropora GFP O A ropora R A ropora K As K ct ct t ropora le le lec ct E E ingle le ingle E S E S

Pooling Condition Pooling Condition a 36 Member Therapeutic Knockin Constructs Library

1. Immune Checkpoints 2. Apoptosis - tPD1 - tBTLA - tFAS - PD1-41BB - BTLA-CD28 - FAS-CD28 - PD1-MYD88 - tTIM3 - FAS-41BB - PD1-ICOS - TIM3-CD28 - FAS-MYD88 - tCTLA4 - tTIGIT - FAS-ICOS - CTLA4-CD28 - TIGIT-CD28 - tTRAILR2 - tCD200R -TRAILR2-CD28

8. Controls - GFP 3. Proliferation - RFP - IL2RA - tNGFR - IL7RA - 41BB

7. Suppressive Cytokines 4. Chemokines - dnTGFβR2 - CCR10 - TGFβR2-41BB - TGFβR2-MYD88 - tIL10RA - IL10RA-IL7RA 5. Transcription Factors - IL4RA-IL7RA 6. Metabolic Pathways - TCF7 - MCT - SOD1 b c Individual Constructs- Arrayed KI 25 Donor 1 ) Donor 2 +

-1 20 ESO -

Y 15 (N

in

ck 10 no

K 5 % Arra yed Knockin of Each Assay Knockin % 36 Member Therapeutic Individual Library Member and TCR Expression 0 1 B 8 4 8 R A 8 3 8 T 8 S 8 B 8 S 2 8 A A B 0 7 2 B 8 A A A P R B 8 A 2 L 2 I 2 A 2 B 8 R 2 R R B 1 T D1 R 8 Knockin Constructs Library D L M D O L F C β B D R R R F F tPD Y T tF Y C L2 L7 41 C M O G RFPG tBT -CD tTI tTIG-CD -CD-41 -I AI I I T S F -41 Y IL7 IL7 tC 4-CD A T R CCR G 2 L10 - - tN (NY-ESO-1 TCR + Gene X into TCRα) D1-41 A tCD200 L -M T -M tI A A L M3-CD2 AStT R 2 P D1-MPD1-ICOS T TI FASFAS F LR2-CD dn β R P T B TIGI FAS AI F β L4R C R G F L10RI T T G I T Immune Checkpoints Apototic Proliferation Chemokines Transcription Factors Metabolic Suppressive Cytokines Controls

d e Individual Constructs- Arrayed KI Individual Constructs- Arrayed KI 30 20000

R2 = 0.03 ) FI ) M

+ -1 -1 15000 20 ESO ESO - - Y Y (N (N 10000

in ion ck 10 ss no

pre 5000 K Donor 1 % Ex Donor 2 CR T 0 0 1 B 8 4 8 R A 8 3 8 T 8 S 8 B 8 S 2 8 A A B 0 7 2 B 8 A A A P R 2000 2500 3000 B 8 A 2 L 2 I 2 A 2 B 8 R 2 R R B 1 T D1 R 8 D L M D O L F C β B D R R R F F tPD Y T tF Y C L2 L7 C O G RFPG tBT -CD tTI tTIG-CD -CD-41 -I AI I I 41 T M S F -41 Y IL7 IL7 tC 4-CD A T R CCR G 2 L10 - - tN D1-41 A tCD200 L -M T -M tI A A Size of Insert (bp, without Homology Arms) L M3-CD2 AStT R 2 P D1-MPD1-ICOS T TI FASFAS F LR2-CD dn β R P T B TIGI FAS AI F β L4R C R G F L10RI T T G I T Construct a + TCR Stimulation

Sequence Isolate Knockin Specific cDNA Pooled Knockin Barcodes

Modified T Cell Library - TCR Therapeutic Knockin Constructs Library (Input) Stimulation (NY-ESO-1 TCR + Gene X into TCRα)

b d e Individual Constructs- Pooled Knockin 8 4 13.9% 3.9% Observed (Cells) Detected (Barcode- cDNA) 6 3

4 2

2 % of Total Reads 1

4.2% % of Total NY-ESO-1+ Cells R2 = 0.11 NY-ESO-1 RFP 0 0 TCR GFP GFP+ RFP+ 2000 2500 3000 Size of Insert (bp, without Homology Arms) c f Input Library Distribution- Day 7 Post Electroporation Individual Constructs- TCR Stimulation 5 3 Fas Derived

4 2

3 1

2 0 % of Total Reads 1 -1 Log 2 Fold Change vs Input Gini Coefficient = 0.048 0 -2 ) 8 S n n 8 A 0 A 8 8 8 B n S n 8 n 8 8 y n n B 8 n n n A n n n 8 8 y 8 0 1 n n 8 ) 7 2 A d d 8 1 2 8 2 d d 8 d 2 T 2 r d d 2 B B d P d d D1 d 8 n T n n 8 A A B A B d S P 8 n d B 8 A 7 R S 8 n B F R O R R B O d C er B B B F R d d d d d B D B O d B F O d B C IC 4 -7 s C y A h A G O 4 MC A C GF A C GF C s T CD D1 A L-7 L CD CD 41 a I M3 M L CD M CD 41 CD 41 41 L-2 S A CD2 L L-7R CD2 41 CD2 CD2 SO L-7R 41 I CD2D1 41 CD2L-2RT a 41 ( IL-7 1 P L MyD CCR Myd F s C LR2 s bR2I L M3 LR2bR2 Myd8 L-7R CCR1 I MyD8 I ( tN Myd8F D T I A s as TI 2 BT a m 4 a T 4 TI s A P s I s -1 M3 A I A L a F AI A D1 TIGIT A BT AI A I A I a mCherr D1 TIGITD1 L M3 -1 a s a P C D1 F bR2 R F LR2 L-10RR L P F C L LR2 F P P L-10RbR2D1 F a F F TI P BT TIGIT b I T CD200R TGF R TGFbR2CD200R I P TI F F C AI T C T T AI TIGIT BT C T IL-4R -10R TGF R C IL-4R R TGF T IL TGF T TGF IL-10RT Construct Construct g h Technical Replicates- Pooled Electroporation Pooled Assembly v Electroporation Technical Replicates- Pooled Assembly Pooled Knockin- Expansion 2 3 1.5 5

2 1.0 4 1

1 0.5 3 0 0 0.0 2 Replicate 2 Replicate 2 -1 Pooled Electroporation -1 -0.5 1 per 1 million input cells (millions)

R2 = 0.99 R2 = 0.88 R2 = 0.89 Total Number of Live Edited Cells -2 -2 -1.0 0 -2 -1 0 1 2 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Input 4 7 10 Replicate 1 Pooled Assembly Replicate 1 Log 2 Fold Change with Stim over Input Log 2 Fold Change with Stim over Input Log 2 Fold Change with Stim over Input Day Post Electroporation

i j k Bulk v Sorted Cells- No Stim, Day 7 1.5 Individual Constructs- No Stim Day 10 vs Day 4 5 1.5

1.0 IL2RA 4 1.0

0.5 0.5 3 0.0 0.0 2 -0.5 -0.5 Post Electroporation

1 Log 2 Fold Change vs Input -1.0 -1.0 Log 2 Fold Change vs Day 4 ) A 0 y 8 8 8 n A 8 n 8 n n n n n 8 7 n 8 T A 8 P 8 B r 2 2 S R d n d 2 B S n B B 2 A F d 8 C R R 2 F 2 1 B 8 O D1 F R d 8 B d d O d d d d B B R Sorted NY-ESO-1+ % of Reads d er R2 = 0.87 C 4 y M -7 G h C O G A A C A L-7 L CD CD 41 CD CD I S N L-7 L M3 CD 41 I D1 as 41 41 CDL-2 (T L M I I CCR C MyD 1 t bR2 Myd A LR2 P F s 4 I 0 T m as D A I TI as a -1.5 2 A M3 BT as L D1 TIGITF AI F A -1 C R bR2 F D1 LR2P F P L-10R R L 0 1 2 3 4 5 Day 7 Day 10 F b TI TIGIT P AI TGF BT I T CD200R T C -10R L-4R C T L TGF R I Bulk Population- % of Reads Day Post Electroporation TGF I T a

8 /CD2 n tio CD3 + imula St β Stim + TGF Apply Isolate Sequence Selective cDNA Knockin Specific Pooled Knockin Pressure TCR Stim Barcodes Only Modified T Cell Library Therapeutic Knockin Constructs Library (Input) Excessive (NY-ESO-1 TCR + Gene X into TCRα) Stim b Individual Constructs- TGF-β vs Input Individual Constructs- TGF-β vs Stim Only 2 2 Fas Derived TGF-βR2 Derived 1 1

0 0

-1 -1 Log 2 Fold Change vs Input

-2 -2 A 0 8 n n y n ) n 8 8 n n 8 8 A 1 d A B P n 8 B S d r d R n T 8 8 7 n A 8 S B n B 8 n 8 8 8 A 8 n 0 8 n n 1 8 A n n y A n n 8 T ) n 8 A n d 2 2 d d 2 2 R R 2 R B F d 8 B O D1 F d C 2 8 F d R 8 B d B d d d d D d d d d 7 d d er O Log2 Fold Change vs CD3/CD28 Stim BB BB 4 A G D C O A h d s BB OS OS BB F 4 d8 L CD CD CD D1 -7 41 y 41 I G M CD y C IC 41 a 41 C GFR A A GFPC MCC y A CD CD M3 IL-7 L-7CCR L M S C N M (T IL-2 Myd F as 41 I N CD2CD2 CD2L-7R L CD241 D1 I SO CD2L-7R 41 L-7RM3 CD2 T A L-2R 41 L 4 A P I LR2 1 m t bR2 Myd8F t I MyD8 CCR14 P LR2 ( L M I bR2 T TI BT as L A I A D1 D TIGIT 2 as as as as A as BT A I TIGITmCherr TI T A F LR2 AI D1 P P L-10R M3R -1 as F F as F L A D1 AI D1 A I M3 -1 C C L R I CD200RTI b F F bR2 F F F D1 L L-10R P LR2 R P bR2 T TIGIT BT AI T P C TGF TIGITBT P T I T CD200RTI F bR2 TGF C L-4R R -10R T C AI C I L TGF R IL-4R T TGF T I TGF T IL-10R TGF Construct Construct

c Individual Constructs- TCR Stim (without Co-stim) vs Input Individual Constructs- TCR Stim (without Co-stim) vs Stim Only 2 Fas Derived 2 CD28 Switch Receptors

1 1

0 0

-1 -1 Log 2 Fold Change vs Input

-2 -2 n n 8 0 n 8 n A 8 y A n T n 1 B 8 S n ) 8 n n n 8 B 8 8 8 n B 8 n n 8 A n n n y B A A 1 0 S 8 8 8 8 B B n n ) n A n 8 8 8 d A d d d B P d d d 7 B R d A S d S d d R d d d P D d d 7 d d

d Log2 Fold Change vs CD3/CD28 Stim d8 B D B O F B d O B s B O B O B B F 4 A y GF MC A yD8 C C C a C GF A A GF C 4 C MCT A L CD2 D1 CD2 L-7R 41 SO 41 I T CD241 GF as 41 CD2 CD2 CD2 41 D1 CD2 L-7RL 41 L-7RL-7RSO I CD2 CD241 41 A T L-2R M3 CD2CD2 CD2 L L-7R CCR1P M LR2 IL-7R bR2 M ( tN F M3 IL-2R Myd8 I s F s Myd8s I P LR2 tN I CCR1 Myd8MyD8 L bR2( I mCherr TI a A 4 a s a BT s T TI A 4 T A I BT AI A I D1 D1 -1 as TIGIT as as L A M3 F a AI A I mCherrD1 A I D1 a -1 TIGIT L M3 A C LR2 P L-10R bR2D1 P F F F F L F F R LR2 L-10R P P D1 F bR2C F L bR2R TIGIT TGF I P CD200R F BT T TI T I CD200R TIGITbR2 P TGF BT TI T AI T C C AI C C R IL-4R TGF T R IL-4R TGF T IL-10R T TGF T IL-10R TGF Construct Construct

d Individual Constructs- Excessive Stim (5:1) vs Input Individual Constructs- Excessive Stim (5:1) vs Stim Only 2 Fas Derived 1.0 TCF7 Derived 0.5 1

0.0

0 -0.5 Log 2 Fold Change vs Input -1 -1.0 8 n 8 8 n A 0 S B n n n 8 A 8 1 B n 8 n A y n 8 8 ) S 8 n B B 8 n 8 A n 8 8 8 8 A 0 1 n A A 8 n n n n n y 8 8 ) d d A d B d d D d T d P d R 7 d R d S S B d B d n d d P d d B d T 7 O B B B d8 F O s B B O O B B D d B d8 F A C D8 GF A y GF C C a Log2 Fold Change vs CD3/CD28 Stim GF s C C A A GF y MC CD2M3 CD2CD2L L-7R I 41 41 D1 CD2L-7R y SO 41 MC CD2 L-2R CD2 T 41 41 CD2a I 41 D1 CD2 CD2CD2 41 SO L-7RL L-7RCD2 M3 41 CD2 C 4 L-7RCCR1 bR2 P LR2 I M I M tN ( s I Myd8F s s tN F Myd8s I IL-2RP MyD8 IL-7RCCR1 bR2 LR2 M (T TI A BT as TIGIT mCherr a s a a A s a 4 s TI mCherr A L A I A I D1 AI D1 M3 -1 F a F F L a D1 M3 A A I BT A I a TIGIT AI D1 -1 L P bR2 R F D1 P L-10R LR2 F F F F P bR2 D1 L L-10R F P LR2 T BT TIGIT TGF T P CD200R TI I bR2 BT TI P T TIGIT I TGF R CD200R bR2 F C AI C C T AI C IL-4R TGF R T TGF IL-4R R T IL-10R TGFT IL-10R T TGF Construct Construct a

Sequence Isolate Knockin cDNA Specific Pooled Apply Dissect Barcodes Knockin Selective Tumor Pressure Sorted Modified In Vivo T Cells T Cell Library Solid Tumor (Input) Xenographt Model Therapeutic Knockin Constructs Library (NY-ESO-1 TCR + Gene X into TCRα) b c d Biologic Replicates Technical Replicates 1.0 TGF-βR2 Derived 1.0 TCF7

1.0 t u 0.5 np I

0.5 - 0.5 e g

n 0.0 a 2 2

0.0 h r e C o s

u 0.0 on o

old -0.5 D

-0.5 M F 2

-0.5 -1.0 Log -1.0

R2 = 0.160 R2 = 0.157 -1.5 -1.0 -1.5 n n n n B 8 n 8 8 8 0 y n S n n B n 8 8 8 A A n B 8 ) A A B 8 d S d d 2 d 8 P 2 1 r d d d d 2 8 2 d T 8 7 -1.5 -1.0 -0.5 0.0 0.5 1.0 -2 -1 0 1 d R R B 2 D1 O B 2 F er O B R R C B F A O 4 C s G h A C d L -7 41 S A I a 41 CD CD CD I D1 41 CD CD M 41 y C L IL-2 CD L F M3 s s MyD CCRC P LR2 Myd L-7IL-7bR2 M T I A T 1 TI a a 4 m as s ( BT A L D F A F AI D1 TIGIT a M3 A I 2 Donor 1 Mouse 1 C P F D1 LR2 L L-10R R P F bR2R -1 CD200RP T I T TIGIT TI TGF b F BT AI C C -10R R IL-4R TGF T IL T TGF Construct b 2 PD1 CD39 Lag3 100 100 80 y

a nl Stim + TGFβ e 80 v O i 90 70 t ion t osi 60 Expansion 80 60 s P e v i

imula 40 t 1

TCR β TGFβR2-41BB TCR α VJ Cell Stim 70 50 % 20 Rela vs St

0 60 40 β α 0 2 4 6 TGFβ: - + - + - + - + - + - + 1 2 2 1

R R R R TGFβR1/2 Day post re-stimulation Control (GFP) TGFBR2-41BB Extracellular c 100000 + TGFβ Cancer Cells Only - TGFβ + TGFβ 60 Control (tNGFR) Control (tNGFR) 40

t TGFBR2dn TGFBR2dn 41BB (+ TGFβR1) TGFBR2-41BB + TGFBR2-41BB s Coun D1

Intracellular 30 40 10000 Hour s P

New Specificity Cell + New Function (TCR, NY-ESO-1) 96 20 (Anti-Suppressive, t Cell a

TGFβR2-41BB) 20 % Cancer 10 1000 0 0 T cell to Cancer Cell Ratio: 4:1 2:1 1:1 1:2 1:4 4:1 2:1 1:1 1:2 1:4 4:1 2:1 1:1 1:2 1:4

e PD1 CD39 Lag3 5 ) 50 100 80 Stim e GFP 4 ( 40 90 iv d t 60 i rol s t

3 o 30 80 Con

s P 40 Expansion 2 20

ive 70 t BB v Cell TCR β FAS-41BB TCR α VJ 20 -41 %

Rela 1 10 60 No Stim FAS 0 0 50 0 - - - β α 0 2 4 6 Stim: - + - + - + + + + FAS Day post restimulation Control (GFP) FAS-41BB Extracellular f 100000 Cancer Cells Only

g 1.5 s in Control (GFP)a r s e s k r FAS-41BB es r a 41BB ) l p P M x

Hour Intracellular F E

e

1.0 Cel G

s ( l

l ac 96 f

er 10000 e ol r New Specificity New Function t c r u t a + C

n S (TCR, NY-ESO-1) t (Anti-Apoptotic, o % d

Can C e

FAS-41BB) e t 0.5 s v i a PD1 t v Coun c i a l CD39 e nd I R LAG3 1000 0.0 T cell to Cancer Cell Ratio: 4:1 2:1 1:1 1:2 1:4 4:1 2:1 1:1 1:2 1:4

PD1 CD39 Lag3 h 1.5 100 100 High Stim 100 ) 80

e 80 g GFP 90 ( iv

1.0 t rol Stim osi t 60 60 P

Expansion 80 Con ive Cells

t 40 v 0.5 40 7 % F C TCR β TCF7 TCR α VJ Rela 70

T 20 20

β α 0.0 0 60 0 0 2 4 6 Stim: + +++ + +++ + +++ + +++ + +++ + +++ i Day post restimulation Control (GFP) TCF7 100000 Cancer Cells Only s 1.5

Control (GFP) g s in Hour r

s

TCF7 e s k 96 r e r t TCF7 a ) p a P M x

t 1.0 Expression F E e

G c s ( l

l

10000 a f e ol r r Coun u t C

n S

New Specificity New Function o % d + C Cell e

e

(TCR, NY-ESO-1) (Altered Transcription, t 0.5 s v i a t v c i TCF7) a PD1 l e nd

I CD39 R

Cancer LAG3 1000 0.0 T cell to Cancer Cell Ratio: 4:1 2:1 1:1 1:2 1:4 4:1 2:1 1:1 1:2 1:4