Supplementary Figure 2: Complete list of intermediate and final LADL plasmids.

(a) Plasmid S13.1. Backbone plasmid used to create the Anchor plasmids. Ampicillin resistant. (b) LADL Anchor plasmid with the dCas9-CIBN. CRY2/CIBN heterodimerization based Anchor plasmid with puromycin resistance. The relevant parts are: EF1A promoter (EF1α); three copies of the FLAG tag (3XFLAG); α – importin Nuclear Localization Signal (NLS); Cryptochrome 2 photolyase homology region (CRY2PHR); SV40 Nuclear Localization Signal (NLS); Glycine-Serine Linker (GS); SV40 Nuclear Localization Signal (NLS); dCas9 D10A H840A; Glycine-Serine Linker (GS); α – importin Nuclear Localization Signal (NLS); Cryptochrome 2 photolyase homology region (CRY2PHR); SV40 Nuclear Localization Signal (NLS); 2A self-cleaving peptide (2A); puromycin resistance (Puro). This plasmid was used in Figures 1–5 and Supplementary Figures 1, 4–11, 13 and is referred to as the “LADL Anchor” plasmid. (c) Empty anchor control plasmid. Puromycin resistance gene expressed from the EF1a promoter without any dCas9-CIBN Anchor protein. This plasmid was used as the puromycin resistant plasmid in the ‘Empty anchor control’ in Figures 1–2 and Supplementary Figures 1, 5, 7–10. (d) Plasmid S12.1. The modified Yamamoto Ampicillin resistant plasmid (Addgene #58768) capable of cloning a single gRNA when cut with BbsI and multiplexing four gRNAs after Golden Gate assembly with the companion B1, B2, B3 Spectinomycin resistant plasmids. This plasmid was used as a backbone to clone gRNA 129 shown in (e). (e) Single gRNA 129 cloned into the Ampicillin resistant S12.1 plasmid backbone using BbsI digestion. (f-h) Single gRNAs 135, 115 and 117 indicated were cloned into Spectinomycin resistant Yamamoto B1 (Addgene # 58778), B2 (Addgene # 58779), B3 (Addgene # 58780) plasmids respectively using BbsI digestion (Supplementary Table 4). The multiplexed assembly is shown in (i). (i) Empty bridge control plasmid. Multiplexed plasmid with gRNAs 129, 135, 115, 117 (Supplementary Table 5). No soluble CRY2 Bridge is expressed from this plasmid, so it is referred to as the “Empty bridge control” plasmid. This plasmid was used in the “Empty bridge control” condition in Figure 1, 5 and Supplementary Figures 5, 13. (j) CRY2olig-mCherry plasmid with BbsI sites mutated. Plasmid used to PCR amplify the mCherry cassette for use in gRNA cloning. The original CRY2olig plasmid Addgene #60032 was mutated at two BbsI sites to give the CRY2olig-mCherry-mut2–1. Kanamycin resistant. (k) Empty target control plasmid. Soluble CRY2 (CRY2HA-2A-mCherry) cassette cloned into the plasmid S13.1. This plasmid was also used to facilitate insertion of CRY2 into the multiplexed plasmids without soluble CRY2. Schematic shown in Figure 1c. As this plasmid lacks the gRNA expression module it is referred to as the “Empty target control” plasmid and was used in Figures 1, 3–5 and Supplementary Figure 4, 6–11 and 13. (l) LADL Bridge + Target plasmid. Multiplexed plasmid with soluble CRY2 and gRNAs 129, 135, 115, 117 all targeting desert regions (Supplementary Table 6). Schematic shown in Figure 1c and plasmid used in Figures 1–5 and Supplementary Figures 1, 5–11 and 13. (m) LADL Bridge + Promoter Only Target plasmid. Multiplexed plasmid with gRNAs 115 and 117 targeting the Zfp462 promoter region with soluble CRY2 (Supplementary Table 6). This plasmid was used in the “One-sided guide control” condition in Supplementary Figures 7–10.