Fig. 4: Silencer model for LMNB1 overexpression in ADLD and generation of CRISPR/Cas9 mediated genomic deletions.

a Model 1—A silencer element acts to maintain low LMNB1 expression in oligodendrocytes (OLs). The silencer-LMNB1 interaction is disrupted in ADLD-causing variants but not in LN-Dup cases. Model 2—Based on a previous study of an ADLD-Del patient, proposes that the upstream deletion causes loss of a TAD boundary bringing an enhancer closer to LMNB1 leading to overexpression⁷. However, it is unclear how this would explain why there is no disease due to LN-Dups. b UCSC genome browser view showing the syntenic ADLD-Crit 1 region in the mouse genome (134kb-Del, grey bar) deleted by CRISPR/Cas9. c Dual guide RNAs (G1 and G2) were cloned into a CRISPR/Cas9 plasmid followed by transfection and FACS sorting for GFP+ single cells. Deletion positive clones were identified by PCR screening using primers that amplify across the deletion junction. Only clones with deletions (+) will show a PCR product. d Representative DNA copy number analysis from positive Oli-neu clone with deletion using real time PCR demonstrates reduced copy number compared to control clone using primers within deleted region (red). n = 3 technical replicates for each clone, ***p < 0.001, two-tailed t-tests. Primers outside deleted region (blue and green) show copy number similar to control cells. e Sequencing deletion junctions using the junction PCR primers shown in (b) reveals each Oli-neu clone has a unique sequence due to imperfect repair after CRISPR/Cas9 mediated deletions. Protospacer Adjacent Motif (PAM) sites are highlighted in blue. Note that PAM sites can be located on the reverse strand, with their orientation indicated by arrows. f Lmnb1 mRNA expression as measured by real time PCR relative to βActin (Actb) is significantly higher in Oli-neu cells with the deletion but is not significantly altered in N2A cells and reduced in 3T3 cells, compared to control cell lines. Oli-neu, control n = 3, del n = 4; N2A, control & del n = 3; 3T3, control n = 5, del n = 4, *p < 0.05, two-tailed t-tests. In all cases, independent clones were used. For all graphs, data are presented as mean values +/− SEM.