Journal: Cell Genomics

Article Title: Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing

doi: 10.1016/j.xgen.2025.100770

Figure Lengend Snippet:

Article Snippet: The gRNA expression plasmid (Addgene #232183) for the single gRNA screens was generated by modifying Addgene #83925 to contain an optimized gRNA scaffold, an mCherry transgene in place of GFP, and a puromycin resistance gene in place of Bsr.

Techniques: DNA Methylation Assay, Sequencing, Recombinant, Plasmid Preparation, Expressing, Software

Journal: bioRxiv

Article Title: RAPID-DASH: Single-Day Assembly of Guide RNA Arrays for Multiplexed CRISPR-Cas9 Applications

doi: 10.1101/2025.04.09.648054

Figure Lengend Snippet: a, Overview of RAPID-DASH. gRNA units are assembled using PCA with unique primer sets that add Type IIS restriction enzyme recognition sites and 4 bp sequences that, when digested by BsaI, enable orderly assembly via Golden Gate assembly. The order of the assembly in this overview is determined by the color-coded Type IIS restriction site flanking the gRNA expression units. b, Screening bacterial clones by digesting out the assembled arrays. Gel electrophoresis image shows bands at ∼4kb, which is the expected size for 10 gRNA arrays. c,d, Bar plot showing the number of gRNAs assembled in array from (c) a single clone whole plasmid and (d) bulk plasmid sequencing. Highlighted bar shows 10 gRNA arrays. e, RAPID-DASH timeline.

Article Snippet: Plasmids isolated from white colonies were screened for correct gRNA array assembly via BsmB1-v2 digestion (NEB R0739, Manufacturer’s protocol).

Techniques: Expressing, Clone Assay, Nucleic Acid Electrophoresis, Plasmid Preparation, Sequencing

Journal: bioRxiv

Article Title: RAPID-DASH: Single-Day Assembly of Guide RNA Arrays for Multiplexed CRISPR-Cas9 Applications

doi: 10.1101/2025.04.09.648054

Figure Lengend Snippet: Functional validation of the assembled arrays. a, GFP reporter assay for gRNA array validation. b, Bar chart showing the percentage of the GFP reporter cells activated by gRNA arrays with GFP-targeting gRNA at each position within the array. NTC=non-targeting control with no GFP-targeting guide c, Abundance (colour) of each guide RNA (gRNA) sequences (y axis) across different positions in an array (x axis) when using a pooled spacer sequences for each array position.

Article Snippet: Plasmids isolated from white colonies were screened for correct gRNA array assembly via BsmB1-v2 digestion (NEB R0739, Manufacturer’s protocol).

Techniques: Functional Assay, Reporter Assay, Control

Journal: Scientific Reports

Article Title: Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy

doi: 10.1038/s41598-020-74772-9

Figure Lengend Snippet: Screening of gRNAs targeting B2M or α chains of HLA-II genes. Raji cells were transfected with the indicated gRNAs complexed with Cas9 protein (gRNA/Cas9) and cultured for 7 days. ( A) Target deletion efficiency of the indicated gRNAs as detected by flow cytometry in Raji cells. Percentages of target HLA-negative cells out of total cells are presented as % HLA neg (n = 3). Error bars indicate mean ± standard error of the mean (SEM). ( B ) Amount of HLA-targeted gene disruption measured by a mismatch-sensitive enzyme assay on DNA amplified from target-gRNA/Cas9- or non-targeting gRNA/Cas9-transfected Raji cells. Expected sizes of the DNA fragments are specified in separate tables below. Representative images are shown from three independent experiments. Full-length gels are presented in Supplementary Figure S3. M, DNA Marker. ( C ) On-target and off-target mutagenesis measured by NGS on genomic DNA amplified from indicated gRNA/Cas9-transfected Raji cells (n = 3). Error bars indicate mean ± SEM. On, on-target. Off, off-target.

Article Snippet: A total of 7.5 μg of Cas9 protein (Toolgen, TGEN_CP1; gRNA screening experiment in Supplementary figure , Clontech, 632640; all other experiments) and 7.5 μg of gRNA per reaction were mixed and incubated at room temperature for 10 min to form the Cas9/gRNA complex.

Techniques: Transfection, Cell Culture, Flow Cytometry, Disruption, Enzymatic Assay, Amplification, Marker, Mutagenesis

Journal: Scientific Reports

Article Title: Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy

doi: 10.1038/s41598-020-74772-9

Figure Lengend Snippet: Efficient HLA disruption in human T cells by multiplex genome editing. Primary human CD3 + T cells were transfected with a mixture of gRNAs targeting B2M, HLA-DRA, HLA-DQA and HLA-DPA (Quadruple gRNAs) or non-targeting gRNA (control gRNA) together with Cas9 protein and expanded for 13 days ex vivo. ( A ) Representative histograms of HLA expression on CD4 + (left) or CD8 + (right) T cells gated from 13-day expanded CD3 + T cells. ( B ) Quantification of HLA expression in CD4 + (top) and CD8 + (bottom) T cells in ( A ) (pooled results from three independently performed experiments, total n = 6). Percentages of target HLA-positive cells out of total cells are presented as % HLA pos. Error bars indicate mean ± SEM. *, p = 0.0313, by Wilcoxon matched-pairs signed rank test.

Article Snippet: A total of 7.5 μg of Cas9 protein (Toolgen, TGEN_CP1; gRNA screening experiment in Supplementary figure , Clontech, 632640; all other experiments) and 7.5 μg of gRNA per reaction were mixed and incubated at room temperature for 10 min to form the Cas9/gRNA complex.

Techniques: Disruption, Multiplex Assay, Transfection, Control, Ex Vivo, Expressing

Journal: Scientific Reports

Article Title: Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy

doi: 10.1038/s41598-020-74772-9

Figure Lengend Snippet: HLA-I/II-negative human T cells retain functional and phenotypic properties. Primary CD3 + T cells were transfected with a mixture of gRNAs targeting B2M, HLA-DRA, HLA-DQA and HLA-DPA (Quadruple gRNAs) or non-targeting gRNA (control gRNA) together with Cas9 protein and expanded for 14 days ex vivo. ( A ) A flow cytometry image of HLA ablation in CD3 + T cells transfected with HLA gRNAs. A representative plot is shown from five different donors. Boxes on the plots indicate the HLA-positive control T cells (blue) or HLA-I/II-negative T cells (red) analyzed in ( B – E ). ( B ) Distribution of CD4 + and CD8 + T cells in HLA positive control T cells or HLA-I/II-negative T cells gated from ( A ). Results from three different donors are shown. ( C ) Histograms depicting expression of activation or exhaustion markers in HLA-positive control T cells (top) or HLA-I/II-negative T cells (bottom) gated from ( A ). A representative image from five different donors is shown. ( D , E ) Secretion of TNF-α, IFN-γ and CD107a release were measured by intracellular staining in HLA-positive control T cells or HLA-I/II-negative T cells gated from ( A ). Cells were stimulated with PMA/ionomycin ( D ) or Dynabeads Human T-Activator CD3/CD28 ( E ). Results from six ( D ) or three ( E ) different PBMC donors are shown. n.s, not significant ( p > 0.05 ) by Wilcoxon matched-pairs signed rank test.

Article Snippet: A total of 7.5 μg of Cas9 protein (Toolgen, TGEN_CP1; gRNA screening experiment in Supplementary figure , Clontech, 632640; all other experiments) and 7.5 μg of gRNA per reaction were mixed and incubated at room temperature for 10 min to form the Cas9/gRNA complex.

Techniques: Functional Assay, Transfection, Control, Ex Vivo, Flow Cytometry, Positive Control, Expressing, Activation Assay, Staining

Journal: Scientific Reports

Article Title: Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy

doi: 10.1038/s41598-020-74772-9

Figure Lengend Snippet: HLA-II ablation in HLA-I-negative T cells further reduces allogeneic T cell activation. ( A ) A schematic diagram of experimental design for ( B , C ) Human primary CD3 + T cells isolated from healthy donor PBMC were transfected with non-targeting gRNA (control gRNA), B2M gRNA (B2M-27 gRNA), or a mixture of quadruple gRNAs each targeting B2M, HLA-DRA, HLA-DQA and HLA-DPA (quadruple gRNAs) together with Cas9 protein and expanded ex vivo. On day 14, the expanded T cells were sorted according to their HLA expression and γ-irradiated. Another set of allogeneic human PBMCs stained with CTV was co-cultured with the sorted T cells for 12-h. ( B ) A representative flow cytometry plot depicting the numbers of cytokine-producing allogeneic CTV + CD3 + T cells co-cultured with the HLA + (top), HLA-I − (middle), or HLA-I/II − T cells (bottom), respectively. ( C ) Quantification of IFN-γ (left) or TNF-α-secreting (right) cells among the total allogeneic CD3 + T cells from ( B ) are presented. Each dot represents different alloreactive donor (n = 13). Pooled results from 4 independently performed experiments are shown. * p < 0.05, by Friedman test followed by Dunn’s multiple comparisons test.

Article Snippet: A total of 7.5 μg of Cas9 protein (Toolgen, TGEN_CP1; gRNA screening experiment in Supplementary figure , Clontech, 632640; all other experiments) and 7.5 μg of gRNA per reaction were mixed and incubated at room temperature for 10 min to form the Cas9/gRNA complex.

Techniques: Activation Assay, Isolation, Transfection, Control, Ex Vivo, Expressing, Irradiation, Staining, Cell Culture, Flow Cytometry

Journal: Genome Biology

Article Title: Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

doi: 10.1186/s13059-021-02263-9

Figure Lengend Snippet: Characteristics of Cas13d-mediated circRNA knockdown. a Optimized Cas13d system targeting 8 circRNAs, each with 2 gRNAs. qRT-PCR for circular and linear transcripts after knockdown of circRNAs in Huh7 cells. b Top, schematic drawing of circEGFP-targeting guide RNA sequence and spacer position-matched shRNA. Bottom, relative circEGFP knockdown by individual position-matched gRNA and shRNA. NT, non-targeting. c Volcano plots of differential transcript levels between circEGFP-targeting and non-targeting shRNAs (left) or circEGFP-targeting CasRx and non-targeting guide (right) as determined by RNA sequencing. d Summary of significant off-target transcript perturbations by matched Cas13d gRNA and shRNA. The data shown are from one of two biological replicates with similar results, and error bars indicating the mean ± s.d. of three technical replicates

Article Snippet: The lentiviral gRNA and pre-gRNA-expressing backbones were constructed by cloning the human U6 promoter and CasRx gRNA or pre-gRNA scaffold (Addgene #109053, #109054) into lentiGuide-Puro (Addgene, #52963) by replacing its original U6-gRNA cassette.

Techniques: Knockdown, Quantitative RT-PCR, Sequencing, shRNA, RNA Sequencing

Journal: Genome Biology

Article Title: Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

doi: 10.1186/s13059-021-02263-9

Figure Lengend Snippet: Systematic comparison of CRISPR-Cas13d and shRNA functional screenings for circRNAs. a Number of gRNAs and shRNAs per circRNA in the library. b Schematic view of the screenings. Cas13d and shRNA lentivirus libraries were infected into CasRx stably expressed Huh7 cells or naive Huh7 cells separately and selected by puromycin treatment (time zero). Puromycin-resistant cells were further cultured for 14 days. Genomic DNA was extracted at indicated time points and library representation was determined by deep sequencing. c Correlation heatmap showing the Pearson correlation coefficient between the levels of gRNAs/shRNAs in biological replicates of time zero samples (D0) and 14-day enrichment samples (D14) for Cas13d screen (left) and shRNA screen (right). d Gene set enrichment analysis (GSEA) revealed essential genes are enriched in negative selections for Cas13d screen (left) and shRNA screen (right). Essential genes serve as positive controls. The degree of enrichment is measured as normalized enrichment score (NES). e Scatterplots showing log 2 -transformed fold-change of gRNA/shRNA normalized read counts in D14 vs. D0 for Cas13d screen (left) and shRNA screen (right). Control, non-targeting controls; Essential, positive controls targeting known essential genes; Target, circRNAs highly expressed in HCC. f Scatterplots showing negatively selected gRNA/shRNAs and corresponding genes from Cas13d screen (left) and shRNA screen (right) with FDR < 0.25. CircRNAs are indicated with genomic locations and the host gene name at the end (e.g., chr10|32197099|32199491|ARHGAP12|). Positive controls only have gene names without genomic location (e.g., EIF4A3). g , h Top, relative expression levels of circZBTB44 and its parental mRNA upon knockdown of circZBTB44 by shRNAs ( g ) and gRNAs ( h ) in human Huh7 cells. Middle and bottom, proliferation rates of control and shRNA-mediated ( g ) or Cas13d-mediated ( h ) circZBTB44-silenced Huh7 cells. The number of cells was detected upon staining with crystal violet, and representative pictures are shown in the middle, while the proliferation curves are shown at the bottom. i, j Top, relative expression levels of circRHOT1 and its parental mRNA upon knockdown of circRHOT1 by gRNAs in Huh7 cells ( i ) or PLC/PRF/5 cells ( j ). Middle and bottom, proliferation rates of control and circRHOT1-silenced Huh7 cells ( i ) or PLC/PRF/5 cells ( j ). The number of cells was detected upon staining with crystal violet. The data shown are from one of two biological replicates with similar results, and error bars indicating the mean ± s.d. of three technical replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired Student’s t test). ns, not significant

Article Snippet: The lentiviral gRNA and pre-gRNA-expressing backbones were constructed by cloning the human U6 promoter and CasRx gRNA or pre-gRNA scaffold (Addgene #109053, #109054) into lentiGuide-Puro (Addgene, #52963) by replacing its original U6-gRNA cassette.

Techniques: Comparison, CRISPR, shRNA, Functional Assay, Infection, Stable Transfection, Cell Culture, Sequencing, Transformation Assay, Control, Expressing, Knockdown, Staining

Journal: Genome Biology

Article Title: Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

doi: 10.1186/s13059-021-02263-9

Figure Lengend Snippet: Larger Cas13d library screening identified sorafenib-resistant circRNAs. a Number of gRNAs per circRNA in the library. b Schematic view of the drug selection screening. Cas13d lentivirus libraries were infected into CasRx stably expressed Huh7 cells then selected with puromycin treatment (time zero). Puromycin-resistant cells were further treated with DMSO (vehicle) or sorafenib for 14 days. Genomic DNA was extracted at day 14, and library representation was determined by deep sequencing. c Plot of fold-change of NT and circRNA gRNAs between DMSO- and sorafenib-treated samples. NT, non-targeting. d Scatterplot showing depletion of specific gRNAs after sorafenib treatment. Color dots indicate gRNAs corresponding with experimentally validated circRNAs as shown in Additional file : Figure S8f, g. e The robust rank aggregation (RRA) scores of top negatively selected circRNAs calculated by MAGeCK. Validated sorafenib-resistant circRNAs are labeled in color dots. CircRNAs are indicated with genomic locations and the host gene symbols at the end (e.g., chr12|112513481|112516545|NAA25|-|). f Relative expression levels of circCNIH4 and its parental mRNA upon knockdown of circCNIH4 by two independent gRNAs. g Knockdown of circCNIH4 sensitized Huh7 cells to sorafenib treatment. Knockdown of circCNIH4 significantly suppressed cell proliferation in the presence of sorafenib. Cell proliferation was measured by crystal violet staining, and absorbance at 590 nm of the treated cells from each group (day 6, day 8, day 10) was normalized to NT of the same day. NT, non-targeting. h Relative expression levels of circFMNL2 and its parental mRNA upon knockdown of circFMNL2 by two independent gRNAs. i Knockdown of circFMNL2 sensitized Huh7 cells to sorafenib treatment. Knockdown of circFMNL2 significantly suppressed cell proliferation in the presence of sorafenib. Cell proliferation was measured by crystal violet staining, and absorbance at 590 nm of the treated cells from each group (day 6, day 8, day 10) was normalized to NT of the same day. NT, non-targeting. j Relative expression levels of circCNIH4 and circFMNL2 in parental and sorafenib-resistant Huh7 cells. k Heatmap display of the relative expression levels of circCNIH4, circFMNL2 and their parental mRNAs upon knockdown of circCNIH4 and circFMNL2 by gRNAs in sorafenib-resistant Huh7 cells. l Knockdown of circCNIH4 or circFMNL2 sensitized sorafenib-resistant Huh7 cells to sorafenib treatment. Heatmap display of the relative cell proliferation of control and Cas13d-mediated circRNA-silenced Huh7 cells treated with either DMSO or sorafenib. Cell proliferation was measured by crystal violet staining, and absorbance at 590 nm of the treated cells from each group (day 6, day 8, day10) was normalized to NT of the same day. NT, non-targeting

Article Snippet: The lentiviral gRNA and pre-gRNA-expressing backbones were constructed by cloning the human U6 promoter and CasRx gRNA or pre-gRNA scaffold (Addgene #109053, #109054) into lentiGuide-Puro (Addgene, #52963) by replacing its original U6-gRNA cassette.

Techniques: Library Screening, Selection, Infection, Stable Transfection, Sequencing, Labeling, Expressing, Knockdown, Staining, Control