Extended Data Fig. 1: Optimization of workflows for base editing in primary human T cells.
From: Mapping variant effects on anti-tumor hallmarks of primary human T cells with base-editing screens
a, Overview of approach for targeted base editing in primary human T cells. b-d, Target sites of sgRNAs against CD2, B2M, and TRBC1/2 sites predicted to generate gene knockout through several mechanisms (SPLd = splice donor site mutation, SPLa = splice acceptor site mutation, SM = start codon mutation, ES = conversion to early stop codon). e, Representative flow cytometry histograms from one human donor showing ABE-mediated knockout of CD2 and B2M using sgRNAs indicated in (b-c), and f, CBE-mediated knockout of CD2, TRBC1/2, and B2M using sgRNAs indicated in (b-d). g, Quantification of base editing efficiency in (e) (n = 3 independent human donors). h, Quantification of base editing efficiency in (f), (n = independent human 4 donors for B2M_ES and TRBC1/2_ES; n = 2 independent human donors for B2M_SPLd and CD2_SM). i, Representative flow cytometry dotplots and histograms demonstrating CBE-mediated knockout of TCRab. For histograms, red indicates gated mTurquoise-negative cells, and blue indicates gated mTurquoise-positive cells. j, Quantification of ABE-mediated knockout of B2M with lentiviral integration of B2M_SM_1 sgRNA and electroporation of ABE mRNA in CD4 and CD8 T cell subsets (n = 2 independent human donors). k, Editing efficiency (measured by % B2M loss on flow cytometry) and viability of T cells transduced with B2M_SM_1 sgRNA and electroporated with varying doses of ABE. Vertical dotted line represents ABE dose selected (per 1e6 T cells) for screens. Error bars represent mean +/− SD (panels g, h, j).
