recombinant dna lrg2 1 addgene 108098 sffv cas9 2a mcherry addgene 251397 sffv krab dcas9 2a mcherry  (Addgene inc)

Journal: Microbial Biotechnology

Article Title: A CRISPRi Gene Regulation System for Bifidobacteria

doi: 10.1111/1751-7915.70260

Figure Lengend Snippet: In vivo validation of functional CRISPR interference in bifidobacteria. (A) Schematic of B. breve UCC2003 strains with integrated CRISPRi and nanoluciferase expression systems. (B) The nanoluciferase reporter is expressed in nanoluciferase containing strains (dCas9‐nLuc) resulting in detectable luminescence as measured by luciferase assay. N = 2, * p < 0.001, two‐way ANOVA. (C) Genetic circuit containing a choline inducible dCas9 (P Bet ) directed to target nanoluciferase. (D) gRNA sequences designed to target the 5′ untranslated region (UTR) of nanoluciferase. The coding sequence is shown in orange, the RBS sequence is highlighted in blue and the dCas9 PAM sequences are underlined in blue ( Sth1 ) and purple ( Spy ). (E) Relative luminescence observed with gRNAs targeting the 5′ UTR of nanoluciferase compared to the non‐targeting gRNA NT1 when dCas9 is expressed at basal (− Choline) or induced (+ Choline) levels. N = 3 for Spy dCas9 and N = 8 for Sth1_dCas9, * p < 0.0001, two‐way ANOVA. Error bars represent standard deviation.

Article Snippet: The coding sequences of catalytically dead Cas9 (dCas9) from Streptococcus pyogenes and Streptococcus thermophilus were codon optimized for expression in B. breve and designed to exclude restriction modification motifs present in B. breve (Bottacini et al. ) prior to being chemically synthesized (Twist Biosciences, San Francisco, CA, US) and cloned into pFREM28 using AatII and XhoI (New England Biolabs).

Techniques: In Vivo, Biomarker Discovery, Functional Assay, CRISPR, Expressing, Luciferase, Sequencing, Standard Deviation

Journal: Microbial Biotechnology

Article Title: A CRISPRi Gene Regulation System for Bifidobacteria

doi: 10.1111/1751-7915.70260

Figure Lengend Snippet: CRISPRi repression of exopolysaccharide production. (A) schematic of the major EPS locus in B. breve UCC2003 and the gRNA target sequences within the Bbr_0430 open reading frame. The dCas9 PAM sequences are underlined. (B) OD measurements (OD 600nm ) of B. breve UCC2003 and a Δ430 mutant strain over an 8‐h time period. The observed drop in OD values for the Δ430 mutant strain is due to cell sedimentation. (C) OD measurements (OD 600nm ) of B. breve UCC2003 and strains containing one of three gRNAs targeting Bbr_0430 . N = 8, * p < 0.05, ** p < 0.01, *** p < 0.005, two‐way ANOVA. Error bars represent standard deviation.

Article Snippet: The coding sequences of catalytically dead Cas9 (dCas9) from Streptococcus pyogenes and Streptococcus thermophilus were codon optimized for expression in B. breve and designed to exclude restriction modification motifs present in B. breve (Bottacini et al. ) prior to being chemically synthesized (Twist Biosciences, San Francisco, CA, US) and cloned into pFREM28 using AatII and XhoI (New England Biolabs).

Techniques: Mutagenesis, Sedimentation, Standard Deviation

Journal: Microbial Biotechnology

Article Title: A CRISPRi Gene Regulation System for Bifidobacteria

doi: 10.1111/1751-7915.70260

Figure Lengend Snippet: CRISPRi repression of fucose metabolism in B. longum subsp. infantis and B. animalis subsp. animalis . (A) Schematic of the operons associated with fucose metabolism in B. longum subsp. infantis ATCC 15697. (B) Growth of B. longum subsp. infantis ATCC 15697 CRISPRi strains with dCas9 alone or in combination with gRNAs targeting fucP in media containing glucose. N = 8 Error bars represent standard deviation (C) Growth of B. infantis ATCC 15697 and CRISPRi strains with dCas9 alone and in combination with gRNAs targeting fucP in media containing fucose. N = 8 Error bars represent standard deviation. (D) Schematic of raffinose operon in B. animalis subsp. animalis ATCC 25527. (E) Growth of B. animalis subsp. animalis ATCC 25527 CRISPRi strains with dCas9 alone or in combination with gRNAs targeting rafA in media containing glucose. N = 13 Error bars represent standard deviation (F) CRISPRi strains with dCas9 alone or in combination with gRNAs targeting rafA in media containing raffinose. N = 13 Error bars represent standard deviation.

Article Snippet: The coding sequences of catalytically dead Cas9 (dCas9) from Streptococcus pyogenes and Streptococcus thermophilus were codon optimized for expression in B. breve and designed to exclude restriction modification motifs present in B. breve (Bottacini et al. ) prior to being chemically synthesized (Twist Biosciences, San Francisco, CA, US) and cloned into pFREM28 using AatII and XhoI (New England Biolabs).

Techniques: Standard Deviation

Journal: Microbial Biotechnology

Article Title: A CRISPRi Gene Regulation System for Bifidobacteria

doi: 10.1111/1751-7915.70260

Figure Lengend Snippet: CRISPRi repression of carbohydrate metabolism in B. longum subsp. longum . (A) Schematic of axu gene cluster in B. longum subsp. longum NCIMB 8809. (B) Growth of B. longum subsp. longum NCIMB 8809 and CRISPRi strains with dCas9 alone or in combination with gRNAs targeting axuA in media containing arabinose. N = 8, error bars represent standard deviation. (C) Growth of B. longum subsp. longum NCIMB 8809 and CRISPRi strains with dCas9 alone or in combination with gRNAs targeting axuA in media containing arabinoxylan from rye. N = 8, error bars represent standard deviation. (D) Growth of B. longum subsp. longum NCIMB 8809 and CRISPRi strains with dCas9 alone or in combination with gRNAs targeting axuA in media containing arabinoxylan from wheat. N = 8, error bars represent standard deviation. (E) Heatmap of genes differentially expressed following CRISPRi targeting of axuA . (F) Top ranked genes differentially expressed in B. longum subsp. longum following CRISPRi targeting of axuA .

Article Snippet: The coding sequences of catalytically dead Cas9 (dCas9) from Streptococcus pyogenes and Streptococcus thermophilus were codon optimized for expression in B. breve and designed to exclude restriction modification motifs present in B. breve (Bottacini et al. ) prior to being chemically synthesized (Twist Biosciences, San Francisco, CA, US) and cloned into pFREM28 using AatII and XhoI (New England Biolabs).

Techniques: Standard Deviation

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

Journal: Nature biotechnology

Article Title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

doi: 10.1038/s41587-025-02853-z

Figure Lengend Snippet: (a) Translation efficiency (log2 (Ribo-seq counts/RNA-seq counts)) negatively correlates with mRNA enrichment in P-bodies in naïve and primed mouse ES cells. Ribosome profiling data from 69 , Pearson correlation test (two sided). (b) A schematic showing CRISPR-Cas9-based homozygous insertion of FKBP12F36V-HA-P2A-mCherry sequence in place of the stop codon of the endogenous Ddx6 allele. (c) Representative intracellular flow cytometry plots for DDX6 in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 at the indicated time points. (d) Representative IF imaging of EDC4 puncta (red) in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 for 6 hours. Nuclei were counterstained with DAPI (blue) (scale: 10μm). (e) P-body number in Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, either untreated (DMSO) or treated with dTAG-13 at the indicated time points. DMSO (n=70 cells), 3 hour-dTAG13 (n=70 cells), 6 hour-dTAG13 (n=70 cells), 9 hour-dTAG13 (n=70 cells), unpaired two-sided Student’s t-test, mean ± s.d., ****: p <0.0001. (f) Cumulative distribution function (CDF) plot showing ribosome occupancy (log2 FC) of P-body enriched and P-body-depleted mRNAs for untreated (DMSO) vs. dTAG-13 treated (6hrs) Ddx6 -FKBP12 F36V GFP-LSM14A mouse naïve ES cells, Wilcox test p=2.97e-129. (g) Box plots showing the change in ribosome occupancy (log2(ribosome bound/total RNA) in the degron-log2(ribosome bound/total RNA) in the control) of P-body enriched genes versus all other genes. Boxes indicate the interquartile range (25th–75th percentile), center lines the median, and whiskers extend to 1.5X IQR. Statistical significance was assessed by unpaired two sided t-test, mean ± s.d., ****: p<0.0001; padj =1.6e-38 (Holm’s method); all other genes n=9133, P-body n=2843. (h) Normalized Enrichment Score (NES) of gene sets from (2C) 64 p=0.003, (Naïve) 105 p=0.923, and (Primed) 70 p=0.499, Enrichment significance was calculated by permutation test (two-sided), with multiple testing correction using the Benjamini–Hochberg method (FDR<0.05). (i) Box plots showing the change in ribosome occupancy of P-body enriched 2C-related genes 64 compared to non-P-body enriched 2C genes. Boxes indicate the interquartile range (25th–75th percentile), center lines the median, and whiskers extend to 1.5 IQR. Unpaired two-sided t-test, mean ± s.d, **: p<0.01; padj=0.044 (Holm’s method); all other genes n=254, P-body n=104. (j) Box plots showing the change in protein levels log2(degron/ctrl) of all P-body enriched genes compared to P-body depleted genes (cytoplasm), after 1 day and 3 days of dTAG-13 treatment. Boxes indicate the interquartile range (25th–75th percentile), center lines the median, and whiskers extend to 1.5X IQR. Unpaired two-sided t-test, mean ± s.d, ****: p<0.0001; padj=8.5e-7, padj=6.6e-6 (Holm’s method); cytoplasm n=1090, P-body n=1661. (k) Box plots showing the change in protein levels log2(degron/ctrl) of P-body enriched 2C-related genes compared to P-body depleted genes (cytoplasm), after 1 day and 3 days of dTAG-13 treatment. Boxes indicate the interquartile range (25th–75th percentile), center lines the median, and whiskers extend to 1.5X IQR. Unpaired two-sided t-test, mean ± s.d, n.s.: p>0.05, *:p<0.05; padj=0.086, padj=0.047 (Holm’s method); cytoplasm n=28, P-body n=42. (l) Heatmap showing protein levels of 2C-related genes after 1 day and 3 days of dTAG-13 treatment compared to control samples.

Article Snippet: Halo-NLS-MCP fragment was PCR amplified from this vector in parallel with PCR amplification of Ires BFP fragment from TRE KRAB Cas9 Ires BFP plasmid (Addgene # 85449) using Phusion polymerase (New England Biolabs) according to the manufacturer’s recommendations.

Techniques: Activation Assay, Gene Expression, RNA Sequencing, CRISPR, Sequencing, Flow Cytometry, Imaging, Control