Journal: Microbial Cell Factories

Article Title: In-situ muconic acid extraction reveals sugar consumption bottleneck in a xylose-utilizing Saccharomyces cerevisiae strain

doi: 10.1186/s12934-021-01594-3

Figure Lengend Snippet: Plasmids used and constructed in this study

Article Snippet: pgRNA-uni-hph (p58) , pBR322 ori ( E. coli ) and 2 micron ori ( S. cerevisiae , multi-copy) gRNA plasmid backbone with hph marker , MCB, KU Leuven.

Techniques: Construct, Plasmid Preparation, Amplification, Marker, Sequencing, Expressing

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

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses

doi: 10.3389/fcimb.2021.590989

Figure Lengend Snippet: Diagram illustrating several structural variants and orthologues of natural Cas9 nuclease. (A) Streptococcus pyogenes Cas9; (B) Staphylococcus aureus Cas9; (C) Neisseria meningitidis Cas9; (D) Sterptococcus thermophilus Cas9; (E) Campylobacter jejuni Cas9; (F) Francisella novicida Cas9; (G) nuclease-dead Cas9; (H) SpCas9 nickase; (I) SpCas9 nickase.

Article Snippet: Nowadays, multiple website platforms are available for the optimal design of CRISPR gRNA (e.g., https://www.genscript.com/gRNA-design-tool.html and https://www.atum.bio/eCommerce/cas9/input ).

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses

doi: 10.3389/fcimb.2021.590989

Figure Lengend Snippet: The illustration showing HIV’s invasion paths in cells and the therapeutic targets of CRISPR/Cas9.

Article Snippet: Nowadays, multiple website platforms are available for the optimal design of CRISPR gRNA (e.g., https://www.genscript.com/gRNA-design-tool.html and https://www.atum.bio/eCommerce/cas9/input ).

Techniques: Biomarker Discovery, CRISPR

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses

doi: 10.3389/fcimb.2021.590989

Figure Lengend Snippet: Applications of CRISPR/Cas9 system for gene therapy of HIV infection.

Article Snippet: Nowadays, multiple website platforms are available for the optimal design of CRISPR gRNA (e.g., https://www.genscript.com/gRNA-design-tool.html and https://www.atum.bio/eCommerce/cas9/input ).

Techniques: CRISPR, Infection, Plasmid Preparation, Transfection, Transduction, TALENs, Cotransfection, Multiplex Assay, Transgenic Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses

doi: 10.3389/fcimb.2021.590989

Figure Lengend Snippet: The life cycle of HBV with editing targets. HBV binds to surface receptors and enters the hepatocytes. Virus particle complete its process of growth and proliferation in host’s hepatocytes. CRISPR/Cas (or CRISPR/Cas9)-mediated disruption of the HBV life cycle can target several loci, which is necessary for the HBV life cycle.

Article Snippet: Nowadays, multiple website platforms are available for the optimal design of CRISPR gRNA (e.g., https://www.genscript.com/gRNA-design-tool.html and https://www.atum.bio/eCommerce/cas9/input ).

Techniques: Virus, CRISPR, Disruption

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses

doi: 10.3389/fcimb.2021.590989

Figure Lengend Snippet: List of CRISPR/Cas9-based antiviral studies on targeting HPV.

Article Snippet: Nowadays, multiple website platforms are available for the optimal design of CRISPR gRNA (e.g., https://www.genscript.com/gRNA-design-tool.html and https://www.atum.bio/eCommerce/cas9/input ).

Techniques: CRISPR, Virus, Transfection, Transduction, Liposomes

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses

doi: 10.3389/fcimb.2021.590989

Figure Lengend Snippet: The illustration shows the potential editing and therapeutic targets in HPV life cycle by the use of CRISPR/Cas and CRISPR/Cas9.

Article Snippet: Nowadays, multiple website platforms are available for the optimal design of CRISPR gRNA (e.g., https://www.genscript.com/gRNA-design-tool.html and https://www.atum.bio/eCommerce/cas9/input ).

Techniques: Biomarker Discovery, CRISPR

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses

doi: 10.3389/fcimb.2021.590989

Figure Lengend Snippet: The schematic diagram showing several challenges of CRISPR/Cas9 in the treatment of human infectious viruses.

Article Snippet: Nowadays, multiple website platforms are available for the optimal design of CRISPR gRNA (e.g., https://www.genscript.com/gRNA-design-tool.html and https://www.atum.bio/eCommerce/cas9/input ).

Techniques: CRISPR

Journal: bioRxiv

Article Title: Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection

doi: 10.1101/2021.06.09.447760

Figure Lengend Snippet: (A) Top: 229E genomic construct map used for the detection of viral genome in fixed cells. 48 FISH probes were designed to target genomic RNA (gRNA) by hybridizing with the RNA-dependent RNA polymerase (RdRp)-coding region, which is only present in the positive-sense gRNA and not in the subgenomic RNAs (sgRNAs). Bottom: Viral genome compared to sgRNAs, which do not contain complementary sequences to the FISH probes. The FISH probes are labeled by CF568 or AF647. Adapted from templates “Discontinuous Transcription” and “Remdesivir Active Molecule Interaction with SARS-CoV-2 RdRp”, by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates . ( Hartenian et al ., 2020 ) (B) Scheme showing double-stranded RNA (dsRNA) targeted by anti-dsRNA antibodies in fixed cells. Each anti-dsRNA antibody recognizes 40 bp of dsRNA. A CF568-labeled secondary antibody is then introduced for visualization. (C) Scheme showing the endoplasmic reticulum (ER) visualized in fixed cells via the expression of a single-pass ER membrane protein, GFP-Sec61B. An AF647-labeled anti-GFP nanobody is used to perform super-resolution (SR) imaging of the ER. (D) Representative confocal images of gRNA (magenta) in GFP-Sec61B-expressing fixed MRC5 cells (gray). Cells were infected with 0.2 MOI 229E and fixed at 6-, 12-, and 24-hour post infection (h p.i.). Nucleus is labeled with DAPI (blue). Scale bar: 10 μm. Right: insets of dotted boxes. Scale bar: 5 μm. (E) Representative confocal images of dsRNA (green) in GFP-Sec61B-expressing fixed MRC5 cells (gray) at 6, 12, and 24 h p.i. with 0.2 MOI 229E. The nucleus is labeled with DAPI (blue). Scale bar: 10 μm. Right: insets of dotted boxes. Scale bar: 5 μm.

Article Snippet: For simultaneous staining of gRNA and ER membrane, transduced cells were incubated with 100 μL Hybridization Buffer containing 2 μL 12.5 μM CF568-labled gRNA FISH probes and 1:2000 AF647-labeled anti-GFP nanobody (Chromotek, gb2AF647–50) for 4 hours in the dark.

Techniques: Construct, Labeling, Expressing, Imaging, Infection

Journal: bioRxiv

Article Title: Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection

doi: 10.1101/2021.06.09.447760

Figure Lengend Snippet: (A) Spatial distribution of fluorescently labeled gRNA in an infected cell via SR microscopy. Numerous nanoscale gRNA puncta, presumably packaged virions, appeared sometimes near the extended clusters. (B) SR reconstruction of isolated virions plated on a coverslip, extracted from cells labeled with the same FISH protocol employed for cellular imaging. Their appearance is very similar to the observed nanoscale gRNA puncta in the cell. Scale bar: 500 nm. (C) Size distribution of purified virions, calculated by fitting the virions with a 2D Gaussian. The mean full width half max of the purified virions was 71 nm. N = 525. (D) DL images of purified virions, immobilized on glass coverslips. FISH probes specifically targeted the viral genome with minimal nonspecific background, as evident from the control condition without virions. Scale bar: 2 μm. (E) Number of FISH probes per virion, calculated by dividing the total fluorescence intensity of individual virions by the single-molecule brightness. On average, each virion is labeled by 18.5 FISH probes. N=441. (F) Plot of virion sizes versus brightness, colored according to the normalized local density of points. There is no clear correlation between the two parameters (Pearson correlation = 0.28). (G) SR reconstructions of nine unique virions stained with spike protein antibodies. The virions are 100–150 nm diameter large bright objects. Scale bar: 100 nm. (H) DL images of two different virions labeled with spike protein (green) and gRNA (magenta). The white color indicates clear colocalization in the center. (I) Two-color SR reconstructions of virions shown in panel H. A distinctive concentric structure of gRNA encapsulated by a larger structure labeled with the spike protein is clearly observed (J) Additional two-color SR reconstructions of several virions where colocalization is evident. 39% of the total virions observed (N=168) exhibited some degree of colocalization. Scale bar: 100 nm. (K) Size distributions of purified virions stained with spike protein antibody. The mean full width half maximum is 119 nm. N=26. (L) Percentage area distribution of gRNA clusters for cells 6 (black), 12 (magenta) and 24 (turquoise) h p.i. Cells infected for a longer period of time showed larger clusters representing increasing copy number of the viral genome. Data collected from 9, 10 and 8 cells, respectively. (M) Cluster density for cells 6 (black), 12 (magenta) and 24 (turquoise) h p.i. Cells infected for a longer period of time exhibited a higher cluster density. *, p< 10 −1 ; **, p< 10 −2 (two-tailed t-test). Data collected from 9, 10 and 8 cells, respectively.

Article Snippet: For simultaneous staining of gRNA and ER membrane, transduced cells were incubated with 100 μL Hybridization Buffer containing 2 μL 12.5 μM CF568-labled gRNA FISH probes and 1:2000 AF647-labeled anti-GFP nanobody (Chromotek, gb2AF647–50) for 4 hours in the dark.

Techniques: Labeling, Infection, Microscopy, Isolation, Imaging, Purification, Fluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection

doi: 10.1101/2021.06.09.447760

Figure Lengend Snippet: (A) Representative confocal images of HCoV-229E-infected MRC5 cells in control and Remdesivir (0.1 μM and 0.5 μM) treated cells using the same brightness and contrast threshold, labeled with FISH probes targeting gRNA (magenta), anti-dsRNA antibody (green) and nuclear staining (blue). Scale bar: 50 μm. (B) Representative confocal images showing gRNA (magenta) and dsRNA (green) in control and 0.1μM Remdesivir-treated MRC5 cells at 24 h p.i. Right: Quantification of the number of dsRNA puncta per cell in control and 0.1μM Remdesivir-treated (RDV) MRC5 cells at 24 h p.i. Scale bar: 10 μm. ****, p< 10 −4 . Data collected from 21 and 25 cells, respectively. (C-D) DL images and corresponding SR reconstructions of two regions of a cell incubated with 0.1 μM Remdesivir at 24 h p.i. where gRNA (magenta) and dsRNA (green) are labeled. dsRNA puncta appeared at the periphery of gRNA clusters, again anticorrelated. (E) Spatial point statistics verify anticorrelation of gRNA and dsRNA. CSR is simulated with the same signal density (black). (F) Sizes of gRNA clusters for Remdesivir-treated cells and untreated cells. Remdesivir treatment reduced the size of the gRNA clusters. *, p< 10 −1 (two-tailed t-test). (G) Sizes of dsRNA puncta for drug-treated cells and untreated cells. **, p< 10 −2 (two-tailed t-test).

Article Snippet: For simultaneous staining of gRNA and ER membrane, transduced cells were incubated with 100 μL Hybridization Buffer containing 2 μL 12.5 μM CF568-labled gRNA FISH probes and 1:2000 AF647-labeled anti-GFP nanobody (Chromotek, gb2AF647–50) for 4 hours in the dark.

Techniques: Infection, Labeling, Staining, Incubation, Two Tailed Test

Journal: Cell reports

Article Title: Reverse engineering neuron-type-specific and type-orthogonal splicing-regulatory networks using diverse cellular transcriptomes

doi: 10.1016/j.celrep.2025.115898

Figure Lengend Snippet: (A) Mean Elavl2 expression RPKM (reads per kb per million) values in MGE- and CGE-lineage interneurons at various time points during cortical development. , , The gene shows a consistent preferential expression in MGE-lineage neurons as early as embryonic day 12.5 (E12.5) and persists into adulthood. (B) Construct designs for the MGE/CGE dual-reporter mouse ESC line. eGFP is positioned downstream of the MGE-specific Lhx6 promoter, and tdTomato is contained in an Ai9 reporter with Cre under the control of the CGE-specific marker 5ht3a . Ascl1/Mash1 overexpression is driven by the neural progenitor marker Nestin to promote interneuron differentiation. (C) Experimental schema for testing the role of Elavl2 in interneuron-type-specific splicing regulation. Elavl2 was knocked out in the dual-reporter mouse ESC line using CRISPR-Cas9. WT and KO ESCs were differentiated into interneurons (ESC-INs) using an embryoid body-based protocol. On day 16 of differentiation, cells were isolated based on reporter fluorescence by FACS and RNA was isolated for RNA-seq. (D) Centered exon inclusion (percent spliced in [PSI]) values of inferred Elavl2 targets ordered by predicted MOR. Inclusion differences of these exons in WT GFP+ (ESC-MGE cells) vs. tdTomato+ (ESC-CGE cells) samples and adult MGE-vs. CGE-lineage interneurons are shown at right. (E) Scatterplots of mean exon inclusion differences (dPSI) in WT versus Elavl2 KO eGFP+ samples ( x axis) compared to WT versus Elavl2 KO tdTomato+ samples ( y axis). Positive and negative Elavl2 regulon exons predicted by MR-AS are indicated by red and blue dots, respectively. (F and G) Overlap (F) and agreement in directionality (G) of DSEs in WT versus Elavl2 KO ESC-MGE cells and Elavl2 regulon exons predicted by MR-AS. (H) Scatterplots of exon inclusion value differences in WT versus Elavl2 KO eGFP+ samples ( x axis) compared to WT eGFP versus WT tdTomato+ samples ( y axis). (I) Agreement in directionality of DSEs in WT versus Elavl2 KO ESC-MGE cells and DSEs in WT ESC-MGE cells versus WT ESC-CGE cells. (J and K) Genome browser views of Slit2 exon 31 (J) and Alcam exon 13 (K) inclusion in WT or KO ESC interneurons and adult MGE- or CGE-lineage neurons. Both exons are inferred targets of Elavl2. p values indicated in (F), (G), and (I) are calculated by Fisher’s exact test.

Article Snippet: Elavl2 knockout synthetic guide RNAs (sgRNA; Synthego multi-guide CRISPR Gene Knockout Kit v2) and Alt-R S.p.

Techniques: Expressing, Construct, Control, Marker, Over Expression, CRISPR, Isolation, Fluorescence, RNA Sequencing