spcas9 plasmid  (Addgene inc)

Journal: Nature

Article Title: Chromosome end protection by RAP1-mediated inhibition of DNA-PK.

doi: 10.1038/s41586-025-08896-1

Figure Lengend Snippet: Fig. 1 | RAP1 and Apollo redundantly prevent cNHEJ at telomeres in mouse and human cells. a, Representative FISH of metaphase spreads of Apollofl/fl Lig4+/+ MEFs 108 h after transduction with single guide RNA (sgRNA) targeting Rap1 (sgRap1) and/or Hit&Run Cre. Telomeres were detected with Cy3-(TTAGGG)3 (green) and DNA was stained with DAPI (magenta). White and green arrows highlight chromatid-type and chromosome-type fusions, respectively. See also Extended Data Fig. 1a. Scale bars 10 µm. b,c, Percentage of telomeres involved in chromatid-type (b) or chromosome-type (c) fusions per metaphase after removal of Apollo and/or RAP1 as indicated, in the presence or absence of LIG4.

Article Snippet: To generate RAP1(KR/DE) clones, 300,000 TP53−/− RPE-1 cells were electroporated with the Neon Transfection System using a 10 μl tip and two pulses at 1,350 V and 20 ms with 500 ng RAP1 guide RNA/ Addgene plasmid #86613, 2 pmol of ATP1A1 ssODN and 6 pmol RAP1 ssODN.

Techniques: Transduction, Staining

Journal: Nature

Article Title: Chromosome end protection by RAP1-mediated inhibition of DNA-PK.

doi: 10.1038/s41586-025-08896-1

Figure Lengend Snippet: Fig. 2 | TRF2, RAP1 and DNA-PK form a terminal complex at telomeric DNA ends. a, Outline of DNase I footprinting experiment. The 32P-labelled 5′ end is highlighted with a red asterisk. Radiolabelled template is incubated with KU and DNA-PKcs prior to the addition of shelterin, comprising TRF1, TRF2, RAP1, TIN2, POT1 and TPP1. DNase I-digested products are then analysed by denaturing

Article Snippet: To generate RAP1(KR/DE) clones, 300,000 TP53−/− RPE-1 cells were electroporated with the Neon Transfection System using a 10 μl tip and two pulses at 1,350 V and 20 ms with 500 ng RAP1 guide RNA/ Addgene plasmid #86613, 2 pmol of ATP1A1 ssODN and 6 pmol RAP1 ssODN.

Techniques: Footprinting, Incubation

Journal: Nature

Article Title: Chromosome end protection by RAP1-mediated inhibition of DNA-PK.

doi: 10.1038/s41586-025-08896-1

Figure Lengend Snippet: Fig. 3 | Three distinct interfaces are required for the complex with DNA-PK. a, Domain organization of RAP1 and TRF2. TRFH, TRF homology domain. b–d, DNase I footprinting of telomere end-binding complexes, testing the requirement for RAP1 RCT or TRF2 RBM (b) TRF2 Myb domain, basic domain or both Myb and basic domains (ΔMΔB) (c), or testing the requirement for TRF2 in the presence of Teb1, RAP1 or Teb1–RAP1 (d). Nucleotides from the 5′ telomeric end indicated. See Extended Data Fig. 2 for details. WT, wild type. e, Protein crosslinking analysis of RAP1 and KU in the presence of DNA. Proteins were mixed with crosslinker and reaction products were separated on a denaturing

Article Snippet: To generate RAP1(KR/DE) clones, 300,000 TP53−/− RPE-1 cells were electroporated with the Neon Transfection System using a 10 μl tip and two pulses at 1,350 V and 20 ms with 500 ng RAP1 guide RNA/ Addgene plasmid #86613, 2 pmol of ATP1A1 ssODN and 6 pmol RAP1 ssODN.

Techniques: Footprinting, Binding Assay

Journal: Nature

Article Title: Chromosome end protection by RAP1-mediated inhibition of DNA-PK.

doi: 10.1038/s41586-025-08896-1

Figure Lengend Snippet: Fig. 4 | Cryo-EM structure of the RAP1–DNA-PK complex. a, Bottom, composite electron density map with protein domains coloured as indicated. Top, schematic of proteins used for structure determination. Uncoloured domains were not visualized. CTD, C-terminal domain; FAT, FRAP–ATM–TRRAP domain; M-HEAT, middle Huntington–EF3–PP2A–TOR1 repeat; N-HEAT, N-terminal Huntington– EF3–PP2A–TOR1 repeat; vWA, von Willebrand A domain. b, Subsection of the structure in a, showing KU70 SAP and RAP1 Myb domains bound to DNA.

Article Snippet: To generate RAP1(KR/DE) clones, 300,000 TP53−/− RPE-1 cells were electroporated with the Neon Transfection System using a 10 μl tip and two pulses at 1,350 V and 20 ms with 500 ng RAP1 guide RNA/ Addgene plasmid #86613, 2 pmol of ATP1A1 ssODN and 6 pmol RAP1 ssODN.

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: Chromosome end protection by RAP1-mediated inhibition of DNA-PK.

doi: 10.1038/s41586-025-08896-1

Figure Lengend Snippet: Fig. 5 | TRF2 and RAP1 prevent cNHEJ by directly blocking recruitment of XRCC4–LIG4 to DNA-PK. a, Outline of the KU pulldown assay. Details in Methods. b–d, KU-bound proteins from reactions containing KU70–KU80 (KU70/80), DNA-PKcs, XRCC4–LIG4, TRF2, RAP1 and template DNA together with wild-type RAP1 (b), RAP1(ΔBRCT) or RAP1(KR/DE) (c), or RAP1(ΔMyb) or RAP1(R133E) (d) were separated by SDS–PAGE and immunoblotted as indicated. TRF2, RAP1 and LIG4 were detected with anti-strep tag antibody, KU70 was detected with anti-Flag antibody. Association of TRF2 with KU is mediated by template DNA. For gel source data see Supplementary Fig. 1. e, Percentage of telomeres per metaphase involved in chromosome fusions upon over-expression of mouse RAP1, RAP1(KR/DE) and RAP1(R130E) (equivalent to human RAP1(R133E)) after CRISPR- and Cre-mediated deletion of Rap1 and Apollo, respectively in Apollofl/fl

Article Snippet: To generate RAP1(KR/DE) clones, 300,000 TP53−/− RPE-1 cells were electroporated with the Neon Transfection System using a 10 μl tip and two pulses at 1,350 V and 20 ms with 500 ng RAP1 guide RNA/ Addgene plasmid #86613, 2 pmol of ATP1A1 ssODN and 6 pmol RAP1 ssODN.

Techniques: Blocking Assay, SDS Page, Strep-tag, Over Expression, CRISPR