DOI: 10.5061/dryad.45j0r70
title: Data from: Reprogramming the antigen specificity of B cells using genome-editing technologies

description: 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.
description: WT_IgM_R1.fastqRamos (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.fastqRamos (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 2 (R2)WT_LLC_R1.fastqRamos (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.fastqRamos (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 2 (R2)M1_IgM_R1.fastqV781-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.fastqV781-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 2 (R2)M1_LLC_R1.fastqV781-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.fastqV781-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.fastqV781-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.fastqV781-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.fastqV781-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.fastqV781-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.fastqV781-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.fastqV781-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 2 (R2)M3_LLC_R1.fastqV781-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.fastqV781-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 2 (R2)W1_IgM_R1.fastqV781-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.fastqV781-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 2 (R2)W1_LLC_R1.fastqV781-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.fastqV781-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 2 (R2)W2_IgM_R1.fastqV781-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.fastqV781-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 2 (R2)W2_LLC_R1.fastqV781-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.fastqV781-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 2 (R2)W3_IgM__R1.fastqV781-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.fastqV781-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 2 (R2)W3_LLC_R1.fastqV781-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.fastqV781-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 2 (R2)M3_RACE_R1.fastqV781-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.fastqV781-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 2 (R2)PU_RACE_R1.fastqV781-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.fastqV781-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 2 (R2)D1_nonengineered_R1.fastqDonor 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.fastqDonor 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 2.D1_V374_PG9_engineered_R1.fastqDonor 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.fastqDonor 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.D2_nonengineered_R1.fastqDonor 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.fastqDonor 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 2 (R2).D2_V374_PG9_engineered_R1.fastqDonor 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.fastqDonor 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 2 (R2).D3_nonengineered_R1.fastqDonor 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.fastqDonor 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 2 (R2).D3_V374_PG9_engineered_R1.fastqDonor 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.fastqDonor 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).