Fig. 5. 5′ and 3′ 12-RSS utilization in V_HQ52.2.4 or V_H7183.4.6-D_H recombination.

(A) Schematic of the 3′ IgH locus CTCF-binding sites (24) and mutations produced by CRISPR-Cas9 in the context of the IGCR1 mutated cell line CBE^−/−(1). (B and D) Rearrangements assays of V_HQ52.2.4 (B) or V_H7183.4.6 (D) to 5′- or 3′-RSS of DQ52 by deletion (orange arrows) or inversion (black arrows), respectively. Locations and orientation of primers used to assay recombination are indicated, together with the 23-RSS of V_HQ52.2.4 (B) or V_H7183.4.6 (D) (blue triangles) and 12-RSSs flanking DQ52 gene segments (green and red triangles). Each set of three lanes contains fivefold increasing amounts of genomic DNA starting at 8 ng (lanes 3, 6, 9, 12, 15, and 18). ROSA26 was used as the loading control. Data shown are representative of two biological replicate experiments. (C and E) Rearrangements assays of V_HQ52.2.4 (B) or V_H7183.4.6 (D) to 5′- or 3′-RSS of DSP2 by deletion or inversion, respectively. ROSA26 was used as the loading control. Data shown are representative of two biological replicate experiments.