ASE function evaluated by CRISPR-Cas9 targeting of the endogenous VL3-3M2 ASE. (A) Nucleotide sequences showing wild-type ASE E2A and GATA3 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), GATA3 (C), and Runx1 (D) binding to the ASE core in wild-type and mutant VL3-3M2 clones. The data represent mean ± SEM of three independent experiments, with enrichment of ASE sequences in specific antibody and nonspecific IgG (control) immunoprecipitates expressed relative to the abundance of Tcra enhancer sequences (set to 1) in specific antibody immunoprecipitates in each cell line. (E) Rag1 and Rag2 transcript abundance assessed by RT-PCR in VL3-3M2 cell clones with wild-type and mutant ASEs. The data represent mean ± SEM of four independent experiments, with values for Rag1 and Rag2 normalized to those for Actb. *, 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) or one-way ANOVA with Holm-Sidak’s multiple comparisons test (E).