Rag1 and Rag2 promoter function evaluated by CRISPR-Cas9 targeting of the endogenous VL3-3M2 Rag1 promoter. (A) Nucleotide sequences showing wild-type Rag1 promoter Ikaros, E2A, and Runx binding sites and mutants generated by CRISPR-Cas9 gene targeting of the two alleles of individual VL3-3M2 clones. Consensus binding sites are highlighted in red. (B–D) ChIP compares E2A (B), Runx1 (C), and Ikaros (D) binding to the Rag1 promoter in wild-type and mutant VL3-3M2 clones. The data represent mean ± SEM of three independent experiments, with enrichment of Rag1 sequences in specific antibody and nonspecific IgG (control) immunoprecipitates expressed relative to the abundance of Tcra enhancer sequences (set to 1) in anti-E2A (B) and anti-Runx1 (C) immunoprecipitates and relative to the abundance of IL2 receptor sequences (set to 1) in anti-Ikaros immunoprecipitates (D). (E) Rag1 and Rag2 transcript abundance assessed by RT-PCR in wild-type and Rag1 promoter mutant VL3-3M2 cells. The data represent mean ± SEM of six to seven independent experiments, with values for Rag1 and Rag2 normalized to those for Actb. (F) Rag1 and Rag2 transcript abundance measured in wild-type and Rag2 promoter–deleted (−119 to +53) VL3-3M2 cells. The data represent the mean ± SEM of five to six independent experiments using three independent ΔRag2p clones, with values expressed as in E. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-way ANOVA with Holm-Sidak’s multiple comparisons test (B–D), one-way ANOVA with Holm-Sidak’s multiple comparisons test (E), or unpaired Student’s t test (F).