Base editing is a highly efficient alternative to nuclease editing. (A) The efficiency of base editing measured by NGS amplicon sequencing in percentage of C-to-T edits at the targeted site is shown on the y-axis. The efficiency at each of 3 sites is demonstrated: β-2 microglobulin (B2M), T-cell receptor α chain (TRAC), and programmed cell death protein 1 (PDCD1). High efficiency is maintained with multiple edits. (*P < .05, all other comparisons not significant). (B) A comparison of CBEs with spCas9. We demonstrate that when 2 or 3 edits are made with CBE, T-cell yield is not impacted (teal bars). In contrast, when spCas9 is used, T-cell yield decreases in a manner proportional to the number of edits made (gold bars). X-axis depicts number of targets edited, and y-axis depicts percentage cell yield as compared with the EP-only condition (*P < .05, **P < .005, all other comparisons not significant). Statistical testing in (A) and (B) was performed using 2-tailed Student’s t-test according to the method of Benjamini, Krieger, and Yekutieli without assuming equal variances between samples. (C) UDiTaS was used to measure translocation frequencies between on-target editing sites in T cells from 1 donor edited at 3 target sites simultaneously. spCas9 induced translocations at on-target sites with frequencies between 0.5% and 1.6%, whereas CBE did not induce detectable translocations. (D) Volcano plots of differentially expressed genes between T cells edited with CBE and Cas9 identified through whole-transcriptome RNA sequencing. Orange circles represent genes that are upregulated and green circles represent genes that are downregulated following editing. Red circles represent genes that are differentially expressed and part of the tumor suppressor TP53 pathway. SpCas9-edited T cells significantly upregulated the TP53 pathway, and CBE does not. The x-axis represents log2 (fold change [FC]), and the y-axis represents P adjusted with the Benjamini-Hochberg method to control for false discovery rate. Significance was determined by an adjusted P value ≤0.05.