Extended Data Fig. 7: dCas9-binding impedes RAG scanning and corresponding loop formation.
From: The fundamental role of chromatin loop extrusion in physiological V(D)J recombination
a, Illustration of the dCas9-block system. An Sγ1-gRNA that has 16 binding sites (blue lines) within a 4-kb highly repetitive Sγ1 region on the C57BL/6 allele was introduced into the JHΔ-dCas9 line. b, Western blot confirmation of dCas9 expression in JHΔ-dCas9 lines but not the parental JHΔ line (done twice with similar results). c, RT–PCR confirmation of Sγ1-gRNA expression in the JHΔ-dCas9-Sγ1-sgRNA lines but not parental lines (done twice with similar results). d, Additional HTGTS V(D)J-seq repeats (DQ52-RSS-DN bait) for JHΔ-dCas9 lines and JHΔ-dCas9-Sγ1-sgRNA lines shown in Fig. 3e. Each library was normalized to the same number of DQ52-RSS-UP CE junctions captured by the DQ52-RSS-DN bait (see Methods). e, Expanded view of Sγ1 region from HTGTS V(D)J-seq profiles in d, showing accumulation of RAG activity at the dCas9-bound Sγ1 region. f, GRO-seq analysis of JHΔ-dCas9 and JHΔ-dCas9-Sγ1-sgRNA lines. Each library was normalized to a coverage of 10 million 100-nt reads for display. Bar graph compares transcriptional activity of indicated regions (n = 3 libraries for each genotype). Data represent mean ± s.d. from biologically independent samples. P values were calculated via two-tailed paired t-test. NS, P ≥ 0.05. The modest decrease in Sγ2b transcription upon Sγ1–dCas9 binding is potentially due to impaired loop extrusion between Sγ2b and iEμ.
