Fig. 1

Utilizing the C-BERST method to target LINE-1 5’UTR. A An APEX2 tagged dCas9 is recruited to the LINE-1 5’UTR promoter using L1Hs specific gRNAs. Cells are incubated with biotin-phenol for 30 min and treated with hydrogen peroxide for 1 min, triggering the biotinylation of proteins located at the LINE-1 5’UTR promoter. Biotinylated proteins are enriched through streptavidin immunoprecipitation and identified through mass spectrometry. Figure and method based on Gao et al. [54]. B Alignment of L1PA2-L1PA7 to L1Hs. Blue lines designate correct alignment to L1Hs, cream lines designate mismatch alignment to L1Hs, and arrowheads mark additional misaligned sequence missing from L1Hs. Eight guide RNAs (gRNA) were designed to target the 5’UTR of LINE-1. Preference went to guides that aligned well with L1Hs, but lacked alignment to older LINE-1 sequences (L1PA2-L1PA7). C Chromatin immunoprecipitation (ChIP) of dCas9 was performed in cells expressing each of the eight LINE-1 5’UTR gRNAs, as well as a non-targeting control (NS). ChIP reads were aligned to LINE-1 L1Hs, as well as older L1PA2-L1PA7, using MapRRCon analysis software [32]. Red line denotes the target location of each gRNA. Representative data shown for gRNA 3, 4, 7 and NS. Complete data set for all gRNAs can be found in Supplemental Figure 1