Fig. 2 | Nature Communications

Fig. 2

From: Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

Fig. 2

Optimization of multiplex editing using optimal sgRNAs (TRAC Ex3 SA, B2M Ex1 SD, and PD-1 Ex1 SD). a Conversion frequency of target cytosine to all other bases at TRAC, PDCD1, and B2M as analyzed by NGS following co-delivery of three target sgRNA with first-generation BE3 or BE4 mRNA; BE4 protein complexed with sgRNA (BE4 RNP); or codon optimized BE4 (coBE4) mRNA. b Indel frequency at TRAC, PDCD1, and B2M as analyzed by NGS following co-delivery of three target sgRNA with first-generation BE3 or BE4 mRNA; BE4 RNP; or coBE4 mRNA. c Indel frequency at TRAC, PDCD1, and B2M as analyzed by NGS following co-delivery of three target sgRNA and SpCas9 nuclease mRNA. d Frequency of TRAC, PD-1, and B2M protein loss measured by flow cytometry seven days post electroporation. e SPICE representation of multiplex flow cytometric analysis performed seven days post electroporation. f Quantification of fractions of WT, single, double, and triple gene KO. Data represented as mean ± SD, n = 2–4 independent biological T-cell donors

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