crincl sublibraries addgene  (Addgene inc)

Journal: Nature Cell Biology

Article Title: Transcription-coupled repair of DNA–protein cross-links depends on CSA and CSB

doi: 10.1038/s41556-024-01391-1

Figure Lengend Snippet: ( a ) Venn diagram and STRING analysis at medium confidence (cutoff = 0.400) showing common hits (resistance or sensitivity) from formaldehyde and 5-aza-dC CRISPRi screens in K562 cells. ( b ) Schematic depiction of formaldehyde detoxification by ADH5 and ESD. ( c ) Schematic showing 5-aza-dC uptake into cells and subsequent phosphorylation events, mediated by DCK and CMPK1, that are necessary for 5-aza-dC incorporation into nascent DNA. ( d-f ) Clonogenic survival assays in WT or CSB −/− TET3G cells in the absence of doxycycline, treated with formaldehyde (d), 5-aza-dC (e) or Illudin S (f); data are presented as mean ± SEM, n = 3 replicates. ( g-h ) Clonogenic survival assays in MRC5 lung fibroblasts and CSB-deficient fibroblasts (CS1AN) treated with formaldehyde (g) or 5-aza-dC (h); data are presented as mean ± SEM, n = 3 replicates. ( i-k ) Alamar blue viability assays in WT, CSB −/− , XPC −/− or CSB −/− / XPC −/− RPE1 cells treated with formaldehyde (i), 5-aza-dC (j) or Illudin S (k); data are presented as mean ± SD, n = 3 replicates. ( l ) Colony formation assay in the cell lines from (i-k) treated with UVC at the indicated doses. ( m ) Representative oligonucleotide excision assay in WT and XPC −/− RPE1 cells treated with formaldehyde (FA) or UVC and released from treatment as indicated. A 50nt oligonucleotide was spiked in as an internal control. ( n ) Quantification of (m); data are presented as mean ± SD, n = 3 replicates. Source numerical data are available in source data.

Article Snippet: A pooled single guide RNA library was generated by combining the human protein-coding genome-wide CRISPRi-v2 library (containing 5 gRNA per gene; Addgene #83969) with multiple CRISPRi non-coding libraries (10 gRNA per gene; Addgene #86538, #86539, #86544, #86548 and #86549), covering the all long non-coding RNA genes expressed in K562 cells.

Techniques: Phospho-proteomics, Colony Assay, Excision Assay, Control

Journal: Nature Cell Biology

Article Title: Transcription-coupled repair of DNA–protein cross-links depends on CSA and CSB

doi: 10.1038/s41556-024-01391-1

Figure Lengend Snippet: a , Schematic of formaldehyde (FA) and 5-aza-dC CRISPRi screens compared to non-treated conditions (NT) in K562 cells. b , Rank plot showing normalized Z-scores (NormZ) scores from DrugZ analysis and selected hits from the formaldehyde CRISPRi screen in a . c , The same as for b but for the 5-aza-dC CRISPRi screen. d , e , Clonogenic survival assays in WT, CSB −/− and XPA −/− HAP1 cells treated with formaldehyde ( d ) or 5-aza-dC ( e ). Error bars ± s.e.m., n = 4 replicates. f , Doxycycline (dox)-inducible expression of CSB or CSB K538R in CSB −/− TET3G HAP1 cells; representative of three independent experiments. g – i , Clonogenic survival assays in WT or CSB −/− TET3G cells with doxycycline-induced expression of CSB, CSB K538R or the empty vector (EV) treated with formaldehyde ( g ), 5-aza-dC ( h ) or illudin S ( i ). Symbols and error bars denote mean ± s.e.m., n = 3 replicates. j – l , Alamar blue cell viability assays in the indicated RPE1 cell lines treated with formaldehyde ( j ), 5-aza-dC ( k ) or illudin S ( l ). Symbols and error bars denote mean ± s.d., n = 3 replicates. Source numerical data and unprocessed blots are available in .

Article Snippet: A pooled single guide RNA library was generated by combining the human protein-coding genome-wide CRISPRi-v2 library (containing 5 gRNA per gene; Addgene #83969) with multiple CRISPRi non-coding libraries (10 gRNA per gene; Addgene #86538, #86539, #86544, #86548 and #86549), covering the all long non-coding RNA genes expressed in K562 cells.

Techniques: Expressing, Plasmid Preparation