EBF1 directly targets an essential binding site in the BENC enhancer region. (A) The sequence of known EBF1-binding sites and 6 predicted EBF1-binding sites within 5 putative Myc 3′enhancer elements as well as a potential EBF1-binding site in the 5′ region of Myc. The core binding site is indicated by a red box. (B) Autoradiogram displaying the result of an electrophoretic mobility shift assay experiment in which the binding of in vitro translated EBF1 to a radioactive labeled Cd79a promoter-EBF1 site is competed for by the addition of a 200-fold excess of nonlabeled putative EBF1-binding sites in Myc enhancers or the PAX5-binding site from the Cd19 promoter. The autoradiogram is representative of 2 independent experiments. (C) Schematic drawing of the targeting of CRISPR guides 106 and 107 to the EBF1-binding site in Myc E2. The DNA sequence of the EBF1 binding motif is depicted in yellow, and guides 106 (light brown) and 107 (dark brown) are shown pointing toward a 3′ NGG PAM sequence. The scale indicates the genomic location on mouse chromosome 15. (D) Myc quantitative reverse transcription PCR data from CD19⁺ iCas9 BM cells transduced with CRISPR guide 81 (control) or 107 (Myc E2) and subsequently treated with doxycycline (DOX) for 48 hours. Mean and standard deviation are shown; n = 6, from 3 individual samples from different mice. (E) Proliferation per 100 iCas9 CD19⁺ BM cells at 24, 48, and 96 hours after DOX administration in samples infected with gRNA constructs sg106 and 107 (targeting EBF1-binding site Myc E2), sg81 (targeting an EBF1 site linked to the Gfra2 gene), or sg112 (targeting the coding region of Myc). Mean and standard deviation are shown. *P < .05; **P < .01, Student t test compared with sg81), from 3 independent samples from different mice.