pcdna krab d sp cas9  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: A split luciferase biosensing platform for detection and imaging of chromatin loops in individual live cells

doi: 10.1093/nar/gkaf1324

Figure Lengend Snippet: Engineering a dCas9-based dual-species biosensor. ( A ) Cartoon of the C-terminal and N-terminal fusion constructs between two orthogonal dCas9 enzymes (d Sp Cas9 and d Sa Cas9) and LgBiT and SmBiT of NanoLuc luciferase. N-terminal LgBit-d Sp Cas9 and SmBit-d Sp Cas9 fusion proteins contain HA epitopes; all other fusion proteins contain the 3× FLAG epitope. All constructs have two nuclear localization signals (NLS). ( B ) Heatmap representing SBR defined as normalized NanoLuc luminescence of on-target sgRNA pairs divided by the normalized NanoLuc luminescence of control “non-loop” sgRNA pairs. Eight fusion protein combinations of the dual dCas9 species DNA biosensor are listed in rows, and 24 different orientations and spacing combinations for MUC4 sgRNA pairings are listed in columns. sgRNA pairs are spaced in tandem, inverted, or everted orientation. Heatmaps show apparent SBRs for each sgRNA pair ( n = 4).

Article Snippet: For d Sa Cas9 fusion constructs, we replaced d Sp Cas9 in pCDNA-KRAB-d Sp Cas9 (Addgene, #112195) with the d Sa Cas9 coding sequence.

Techniques: Construct, Luciferase, FLAG-tag, Control

Journal: Nucleic Acids Research

Article Title: A split luciferase biosensing platform for detection and imaging of chromatin loops in individual live cells

doi: 10.1093/nar/gkaf1324

Figure Lengend Snippet: Biosensing of chromatin loops at the MYC TAD boundaries and at cell-type-specific promoter–super enhancer loops. ( A ) Region capture Micro-C maps of the MYC TAD locus in HCT116 and K562 cells. Cartoon in the center depicts the ∼2.8 Mb MYC TAD. MYC promoter–super enhancer loops targeted with the LgBiT-d Sa Cas9 + d Sp Cas9-SmBiT DNA biosensor are indicated in addition to MYC TAD left (LB) and right (RB) boundary regions. Magenta and green rectangles represent super enhancer regions in HCT116 and K562 cells, respectively. Blue ovals represent all CTCF binding sites outside super enhancer regions. ( B ) Representation of chromatin loop between left and right MYC TAD boundaries. sgRNAs targeting the left boundary are labeled 1–4, and sgRNAs a–d target the right TAD boundary. Heatmap represents chromatin loop biosensing results from 16 pairs of sgRNAs tiling along the pair of convergent CTCF binding sites. Heatmap shows SBR, which is defined as normalized NanoLuc luminescence of sgRNA pairs divided by the normalized NanoLuc luminescence of control “non-loop” sgRNA pairs. (C–E) Cartoon representations of intra-TAD chromatin loops between the MYC promoter and SE regions in HCT116 or K562 cells. Super enhancers 0.53 Mb ( C ) and 8.2 kb ( D ) upstream of the MYC promoter, as well as 1.85 Mb ( E ) downstream of the MYC promoter, were evaluated. sgRNAs were given labels a–f at the MYC promoter binding sites and labels 1–6 at the cell type-specific SE binding sites. Heatmaps summarize chromatin loop biosensing results from 36 pairs of sgRNAs tiling along a CTCF binding site within the large SE and tiling along a highly conserved CTCF binding site at the MYC promoter. Heatmaps show apparent SBRs for each sgRNA pair ( n = 4). Individual SBR scales are shown at top right for each heatmap.

Article Snippet: For d Sa Cas9 fusion constructs, we replaced d Sp Cas9 in pCDNA-KRAB-d Sp Cas9 (Addgene, #112195) with the d Sa Cas9 coding sequence.

Techniques: Binding Assay, Labeling, Control

Journal: Nucleic Acids Research

Article Title: A split luciferase biosensing platform for detection and imaging of chromatin loops in individual live cells

doi: 10.1093/nar/gkaf1324

Figure Lengend Snippet: Imaging MYC promoter-super enhancer loops in live cells using low-light luminescence microscopy. ( A ) Cartoon depicting biosensor targeting the intra-TAD loop between the MYC promoter region and the super enhancer region ∼0.53 Mb upstream from the MYC promoter in HCT116 cells. SE sgRNA 5 paired with MYC promoter sgRNA b is compared to a control “non-loop” sgRNA pair and a NanoLuc-d Sp Cas9 control. Although a single allele is illustrated, luminescence signal is measured per nucleus, not per individual allele. ( B ) Representative live cell microscopy images of split biosensor at chromatin loop between MYC promoter and −0.53Mb SE. GFP fluorescence (green), NanoLuc luminescence (red), and merged images were taken on the Andor Dragonfly 200 Multi-modal Confocal System at 63X magnification. Signal from the MYC promoter and SE sgRNA pair is compared to a control “non-loop” sgRNA pair and a NanoLuc-d Sp Cas9 positive control. Scale bars = 25 μM. ( C ) 25 μM x 25 μM white panels from individual GFP fluorescence (green), NanoLuc luminescence (red), and merged images in panel ( B ) magnified. Scale bars = 3 μM. ( D ) Signal quantification per nucleus (36 h post-transfection) for the same MYC promoter and SE sgRNA pairs using the dual dCas9 species DNA biosensor. Apparent signal-to-background ratio for the ∼0.53 Mb super enhancer sgRNA 5 + MYC promoter b sgRNA pair is listed in parentheses. Unique nuclei were quantified for four conditions: (i) MYC promoter-SE sgRNA pair, (ii) MYC promoter plus non-loop targeting sgRNA, (iii) no sgRNA, and (iv) NanoLuc-d Sp Cas9 ( n = 325, n = 143, n = 9, and n = 193 unique nuclei, respectively). Whisker plots show the median and interquartile range (IQR). Comparisons between group medians were made using an unpaired two-sided Student’s t -test (* P < .05; ** P < .01; *** P < .001).

Article Snippet: For d Sa Cas9 fusion constructs, we replaced d Sp Cas9 in pCDNA-KRAB-d Sp Cas9 (Addgene, #112195) with the d Sa Cas9 coding sequence.

Techniques: Imaging, Microscopy, Control, Fluorescence, Positive Control, Transfection, Whisker Assay

Journal: Nucleic Acids Research

Article Title: A split luciferase biosensing platform for detection and imaging of chromatin loops in individual live cells

doi: 10.1093/nar/gkaf1324

Figure Lengend Snippet: Real-time monitoring of chromatin loop dynamics using an AID system for endogenous RAD21 in HCT116 cells. ( A ) Hi-C contact map at 10 kb resolution showing the MYC TAD in untreated HCT116-RAD21-mAC cells (left) and after 6 h of auxin treatment (right), demonstrating general loss of chromatin loops. The TAD boundary and super enhancer interactions are highlighted with blue and magenta circles, respectively. The intensity of each pixel represents the normalized number of contacts between a pair of loci. Maximum intensity is indicated in the scale at the right. ( B ) Cartoon depicting loss of chromatin loop after inducing RAD21 degradation. ( C ) HCT116-RAD21-mAC cells treated with 1 µM auxin (5-Ph-IAA) for 105 min were compared to untreated HCT116-RAD21-mAC cells over the same 105 min time course. Both treated and untreated conditions were transfected with plasmids expressing LgBiT-d Sa Cas9 + d Sp Cas9-SmBiT and a MYC promoter–super enhancer gRNA pair (gRNA b and gRNA 5, respectively) targeting the −0.53 Mb super enhancer. Untreated and auxin-treated conditions, where no sgRNA pairs were transfected, are shown for comparison. Luminescence was measured ( n = 8) in bulk live cells every 5 min for 105 min for each transfection condition.

Article Snippet: For d Sa Cas9 fusion constructs, we replaced d Sp Cas9 in pCDNA-KRAB-d Sp Cas9 (Addgene, #112195) with the d Sa Cas9 coding sequence.

Techniques: Hi-C, Transfection, Expressing, Comparison