Data from: Reprogramming the antigen specificity of B cells using genome-editing technologies

Voss, James E.1; Gonzalez-Martin, Alicia2; Andrabi, Raiees1; Fuller, Roberta P.1; Murrell, Ben3; McCoy, Laura E.4; Porter, Katelyn1; Huang, Deli1; Li, Wenjuan1; Sok, Devin1; Le, Khoa1; Briney, Bryan1; Chateau, Morgan5; Rogers, Geoffrey5; Hangartner, Lars1; Feeney, Ann J.1; Nemazee, David1; Cannon, Paula5; Burton, Dennis R.1

Published Jan 24, 2019 on Dryad. https://doi.org/10.5061/dryad.45j0r70

Data files

Jan 24, 2019 version files 10.05 GB

D1_nonengineered_R1.fastq.gz

260.03 MB
D1_nonengineered_R2.fastq.gz

234.82 MB
D1_V374_PG9_engineered_R1.fastq.gz

244.07 MB
D1_V374_PG9_engineered_R2.fastq.gz

221.34 MB
D2_nonengineered_R1.fastq.gz

177.39 MB
D2_nonengineered_R2.fastq.gz

156.73 MB
D2_V374_PG9_engineered_R1.fastq.gz

223.91 MB
D2_V374_PG9_engineered_R2.fastq.gz

209.27 MB
D3_nonengineered_R1.fastq.gz

248.93 MB
D3_nonengineered_R2.fastq.gz

221.77 MB
D3_V374_PG9_engineered_R1.fastq.gz

186.74 MB
D3_V374_PG9_engineered_R2.fastq.gz

169.59 MB
M1_IgM_R1.fastq.gz

36.03 MB
M1_IgM_R2.fastq.gz

46.92 MB
M1_LLC_R1.fastq.gz

231.35 MB
M1_LLC_R2.fastq.gz

330.79 MB
M2_IgM_R1.fastq.gz

20.91 MB
M2_IgM_R2.fastq.gz

30.15 MB
M2_LLC_R1.fastq.gz

311.75 MB
M2_LLC_R2.fastq.gz

452.65 MB
M3_IgM_R1.fastq.gz

65.62 MB
M3_IgM_R2.fastq.gz

104.45 MB
M3_LLC_R1.fastq.gz

27.23 MB
M3_LLC_R2.fastq.gz

41.79 MB
M3_RACE_R1.fastq.gz

1.54 GB
M3_RACE_R2.fastq.gz

355.74 MB
PU_RACE_R1.fastq.gz

1.20 GB
PU_RACE_R2.fastq.gz

278.76 MB
W1_IgM_R1.fastq.gz

39.04 MB
W1_IgM_R2.fastq.gz

53.14 MB
W1_LLC_R1.fastq.gz

148.68 MB
W1_LLC_R2.fastq.gz

214.67 MB
W2_IgM_R1.fastq.gz

31.69 MB
W2_IgM_R2.fastq.gz

45.57 MB
W2_LLC_R1.fastq.gz

169.18 MB
W2_LLC_R2.fastq.gz

243.25 MB
W3_IgM__R1.fastq.gz

98.92 MB
W3_IgM_R2.fastq.gz

159.40 MB
W3_LLC_R1.fastq.gz

53.22 MB
W3_LLC_R2.fastq.gz

81.88 MB
WT_IgM_R1.fastq.gz

163.85 MB
WT_IgM_R2.fastq.gz

264.43 MB
WT_LLC_R1.fastq.gz

264.79 MB
WT_LLC_R2.fastq.gz

385.71 MB

Abstract

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody, PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved anti-HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope by FACS and sequenced mRNA from these cells showed PG9 HC expressed as several different isotypes after culture with CD40 ligand and IL-4.

Usage notes

WT_IgM_R1.fastq

Ramos (RA 1) ATCC CRL-1596 Homo sapiens Burkitt's cell lymphoma immunoglobulin heavy chain variable sequences. RT-PCR and amplification from cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

WT_IgM_R2.fastq

WT_LLC_R1.fastq

Ramos (RA 1) ATCC CRL-1596 Homo sapiens Burkitt's cell lymphoma immunoglobulin lambda light chain variable sequences. RT-PCR and amplification from cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

WT_LLC_R2.fastq

M1_IgM_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by one round of selection using HIV MGRM8 SOSIP. Immunoglobulin heavy chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

M1_IgM_R2.fastq

M1_LLC_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by one round of selection using HIV MGRM8 SOSIP. Immunoglobulin lambda light chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

M1_LLC_R2.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by one round of selection using HIV MGRM8 SOSIP.Immunoglobulin lambda light chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 2 (R2)

M2_IgM_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by two rounds of selection using HIV MGRM8 SOSIP. Immunoglobulin heavy chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

M2_IgM_R2.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by two rounds of selection using HIV MGRM8 SOSIP.Immunoglobulin heavy chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 2 (R2)

M2_LLC_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by two rounds of selection using HIV MGRM8 SOSIP. Immunoglobulin lambda light chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

M2_LLC_R2.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by two rounds of selection using HIV MGRM8 SOSIP.Immunoglobulin lambda light chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 2 (R2)

M3_IgM_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by three rounds of selection using HIV MGRM8 SOSIP. Immunoglobulin heavy chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

M3_IgM_R2.fastq

M3_LLC_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by three rounds of selection using HIV MGRM8 SOSIP. Immunoglobulin lambda light chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

M3_LLC_R2.fastq

W1_IgM_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by one round of selection using HIV WITO SOSIP. Immunoglobulin heavy chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

W1_IgM_R2.fastq

W1_LLC_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by one round of selection using HIV WITO SOSIP. Immunoglobulin lambda light chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

W1_LLC_R2.fastq

W2_IgM_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by two rounds of selection using HIV WITO SOSIP. Immunoglobulin heavy chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

W2_IgM_R2.fastq

W2_LLC_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by two rounds of selection using HIV WITO SOSIP. Immunoglobulin lambda light chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

W2_LLC_R2.fastq

W3_IgM__R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by three rounds of selection using HIV WITO SOSIP. Immunoglobulin heavy chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

W3_IgM_R2.fastq

W3_LLC_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by three rounds of selection using HIV WITO SOSIP. Immunoglobulin lambda light chain variable sequences from RT-PCR and amplification of cell mRNA performed using gene specific primers. TruSeq LT amplicon sequenced using 300x300 paired end reads on the Illumina MiSeq. This is read 1 (R1)

W3_LLC_R2.fastq

M3_RACE_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by three rounds of selection using HIV WITO SOSIP. Immunoglobulin heavy and light chain variable regions amplified from cDNA made by RACE with barcoded 5' template switch adapters. NEBnext amplicon sequenced using 400x100 paired end reads on the Illumina MiSeq. This is read 1 (R1)

M3_RACE_R2.fastq

PU_RACE_R1.fastq

V781-PG9 engineered Ramos cells after HIV C108 SOSIP enrichment followed by subsequent passage without further selection, passaged unselected (PU). Immunoglobulin heavy and light chain variable regions amplified from cDNA made by RACE with barcoded 5' template switch adapters. NEBnext amplicon sequenced using 400x100 paired end reads on the Illumina MiSeq. This is read 1 (R1)

PU_RACE_R2.fastq

D1_nonengineered_R1.fastq

Donor 1 control primary B cells 13 days post nucleofection. Immunoglobulin heavy chain variable regions amplified from cDNA made by RACE with barcoded 5' template switch adapters. NEBnext amplicon sequenced using 400x100 paired end reads on the Illumina MiSeq. This is read 1.

D1_nonengineered_R2.fastq

D1_V374_PG9_engineered_R1.fastq

Donor 1, V374-PG9 engineered primary B cells 13 days post nucleofection. Immunoglobulin heavy chain variable regions amplified from cDNA made by RACE with barcoded 5' template switch adapters. NEBnext amplicon sequenced using 400x100 paired end reads on the Illumina MiSeq. This is read 1.

D1_V374_PG9_engineered_R2.fastq

D2_nonengineered_R1.fastq

Donor 2 control primary B cells 13 days post nucleofection. Immunoglobulin heavy chain variable regions amplified from cDNA made by RACE with barcoded 5' template switch adapters. NEBnext amplicon sequenced using 400x100 paired end reads on the Illumina MiSeq. This is read 1.

D2_nonengineered_R2.fastq

D2_V374_PG9_engineered_R1.fastq

Donor 2, V374-PG9 engineered primary B cells 13 days post nucleofection. Immunoglobulin heavy chain variable regions amplified from cDNA made by RACE with barcoded 5' template switch adapters. NEBnext amplicon sequenced using 400x100 paired end reads on the Illumina MiSeq. This is read 1 (R1).

D2_V374_PG9_engineered_R2.fastq

D3_nonengineered_R1.fastq

Donor 3 control primary B cells 13 days post nucleofection. Immunoglobulin heavy chain variable regions amplified from cDNA made by RACE with barcoded 5' template switch adapters. NEBnext amplicon sequenced using 400x100 paired end reads on the Illumina MiSeq. This is read 1 (R1).

D3_nonengineered_R2.fastq

D3_V374_PG9_engineered_R1.fastq

Donor 3, V374-PG9 engineered primary B cells, 13 days post nucleofection. Immunoglobulin heavy chain variable regions amplified from cDNA made by RACE with barcoded 5' template switch adapters. NEBnext amplicon sequenced using 400x100 paired end reads on the Illumina MiSeq. This is read 1 (R1).

D3_V374_PG9_engineered_R2.fastq

Donor 3, V374-PG9 engineered primary B cells 13 days post nucleofection. Immunoglobulin heavy chain variable regions amplified from cDNA made by RACE with barcoded 5' template switch adapters. NEBnext amplicon sequenced using 400x100 paired end reads on the Illumina MiSeq. This is read 2 (R2).