Journal: bioRxiv

Article Title: Dissecting the functions of NIPBL using genome editing: The importance of the N-terminus of NIPBL in transcriptional regulation

doi: 10.1101/616086

Figure Lengend Snippet: 293FT NIPBL mutant cell lines. ( A ) NIPBL gene structure. Exon and intron lengths are not proportional. gRNA target sites within exons 3 and 10 are indicated by arrows. ( B ) Western blotting results for NIPBL. Use of an antibody against the N-terminus (N-ter) or against the C-terminus (C-ter) yielded two bands. The upper band represents full-length NIPBL, and the lower band represents the shorter isoform. Western blotting with clone ex3mut total cellular lysate and the N-ter antibody demonstrated the absence of full-length NIPBL. Western blotting with the C-ter antibody demonstrated the presence of truncated NIPBL. Western blotting of clone ex10mut lysates demonstrated a decreased amount of full-length NIPBL and an increased amount of the truncated NIPBL isoform. ( C ) RNA-seq results for the NIPBL locus. The IGV browser view shows the presence of the NIPBL transcript in clone ex3mut. Transcripts lacked only a few base pairs targeted by NIPBL gRNA. ( D ) Western blotting for MAU2 and SMC1A in the total cellular lysate, soluble fraction (Sup), and chromatin fraction (Chr) in 293FT cell line clones. MAU2 was almost absent in the chromatin fraction of clone ex3mut, and its level was reduced in clone ex10mut.

Article Snippet: The gRNA target sequences GAAGAGGTGAACTGCCTTT ( NIPBL exon 3) and CTCGTTCTGATTTTAACCG ( NIPBL exon 10) were cloned into the gRNA empty vector using Phusion polymerase (M0530S: New England Biolabs, Ipswich, MA) and the Gibson assembly system (E5510S: New England Biolabs).

Techniques: Mutagenesis, Western Blot, RNA Sequencing Assay, Clone Assay

Journal: Developmental cell

Article Title: Maternal ribosomes are sufficient for tissue diversification during embryonic development in C. elegans

doi: 10.1016/j.devcel.2019.01.019

Figure Lengend Snippet: Key Resources Table

Article Snippet: Empty vector for gRNA cloning, pRB1017 , Addgene , 59936.

Techniques: Knock-Out, Recombinant, DNA Library Preparation, DNA Sequencing, Gene Expression, Plasmid Preparation, Cloning, Software, Imaging, Light Microscopy, Fractionation

Journal: Nucleic Acids Research

Article Title: Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

doi: 10.1093/nar/gkw179

Figure Lengend Snippet: HR repair of nicks on the lagging-strand template is promoted by replication. ( A ) The DR-GFP reporter consists of two inactive copies of the GFP gene. Induction of a DSB or nick in SceGFP triggers HR that uses iGFP as a template to restore the GFP open reading frame. The inset shows the SceGFP sequence around the 18 bp I-SceI recognition site, which begins at the in-frame stop codon (red). PAMs required for Cas9 binding are shaded gray and the sequences bound by the indicated gRNAs are shaded in light blue (gRNA01) and light purple (gRNA02). Cas9 variants: wild-type (Cas9 WT ), D10A (Cas9 D ) and H840A (Cas9 H ). The predicted Cas9 cleavage sites are marked by colored triangles. T - transcribed strand, N - non-transcribed strand. ( B–D ) U2OS-DR-GFP cells (B and C) or ES-DR-GFP cells (D) were transfected with expression vectors for the indicated Cas9 and gRNA variants, or I-SceI. As a negative control (first bar in graphs B–D), Cas9 WT was transfected with a gRNA expression vector that lacked the targeting portion of the gRNA sequence. ( E ) U2OS-DR-GFP cells were arrested at the G 2 /M phase transition by 16 h incubation in 500 nM nocodazole or mock-treated with DMSO, stained with propidium iodide and analyzed by flow cytometry to confirm the cell cycle arrest. The left panel shows representative histograms of DNA content. The right panel shows the average percentage of cells in the indicated cell cycle phases. ( F ) U2OS-DR-GFP cells were treated as in (E) and transfected with the indicated Cas9 and gRNA expression vectors, then mock-treated or incubated in the presence of nocodazole for a further 24 h and analyzed by flow cytometry. The cell cycle distribution of cells at the time of flow cytometric analysis is shown in Supplementary Figure S1I. ( G ) Schematic overview of the DR-Orip-GFP reporter. EBNA1 initiates replication downstream from SceGFP , such that the top strand becomes the lagging-strand template. The inset depicts the predicted sites of cleavage by Cas9 D (filled triangles) or Cas9 H (empty triangles), in combination with the indicated gRNAs. ( H ) U2OS cells were transfected with the DR-OriP-GFP reporter, the empty vector (+ Vector) or EBNA1 expression vector (+ EBNA1), gRNA01 and the indicated Cas9 variant. Cas9 D nicks the leading-strand template and Cas9 H nicks the lagging-strand template, as indicated. ( I ) Cells were transfected as in (H), except gRNA02 was used, such that Cas9 D nicks the lagging-strand template and Cas9 H the leading-strand template.

Article Snippet: Cas9 WT , Cas9 D and empty gRNA expression vectors were purchased from Addgene (ID 41815, 41816 and 41824) ( ).

Techniques: Sequencing, Binding Assay, Transfection, Expressing, Negative Control, Plasmid Preparation, Sublimation, Incubation, Staining, Flow Cytometry, Variant Assay

Journal: Nucleic Acids Research

Article Title: Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

doi: 10.1093/nar/gkw179

Figure Lengend Snippet: nick HR requires established HR factors but is not suppressed by NHEJ components. ( A and B ) U2OS-DR-GFP cells transfected with the indicated Cas9, I-SceI and gRNA expression vectors were incubated for 48 h in the presence of the various concentrations of ATM inhibitor KU55933 (A) or ATR inhibitor VE-821 (B). ( C ) Schematic representation of HR and its steps that are inhibited by the BRCA1-interacting peptide hB202 (from BARD1) and the RAD51-interacting peptide BRC3 (from BRCA2). ( D ) U2OS-DR-GFP cells were transfected with the indicated Cas9 and gRNA or I-SceI expression vectors and either with the empty expression vector (pCAGGS) or with the vectors encoding the hB202 and BRC3 peptides. ( E ) Canonical NHEJ is known to suppress DSB HR, but its effect on nick HR is uncertain. ( F ) Wild-type (J1), Ku70 −/− or Xrcc4 −/− ES-DR-GFP cells were transfected with the indicated expression vectors. ( G ) Ku70 −/− ES-DR-GFP cells were transfected with the indicated expression vectors and either the empty (+ Vector) or KU70 (+ KU70) expression vector. ( H ) Wild-type (E14) or Dna-Pk −/− ES-DR-GFP cells were transfected with the indicated expression vectors. ( I ) U2OS-DR-GFP cells were transfected and analyzed as in (A and B), but incubated in the presence of various concentrations of a DNA-PK inhibitor (NU7441).

Article Snippet: Cas9 WT , Cas9 D and empty gRNA expression vectors were purchased from Addgene (ID 41815, 41816 and 41824) ( ).

Techniques: Transfection, Expressing, Incubation, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

doi: 10.1093/nar/gkw179

Figure Lengend Snippet: Paired nicks induce intrachromosomal HR. ( A ) Schematic of paired nicks with the potential to generate 5′ or 3′ overhangs to be tested for induction of PN HR. ( B ) Reporters for measuring PN HR between repeats. The left panel shows a schematic of the pnDR-GFP20-940 bp reporters. The inset shows the relative binding positions of the gRNAs (marked by the green or black horizontal bars) and Cas9 cleavage sites (dotted vertical lines). The lengths of the 5′ or 3′ nick offset as well as the heterology (orange bars) are indicated. The right panel shows expected cleavage positions in the pnDR-GFP reporter with the indicated gRNA/Cas9 combinations. ( C-E ) U2OS cells were transiently transfected with the indicated pnDR-GFP reporter and Cas9/gRNA expression vectors required to generate single or paired nicks or DSBs (schematically drawn under each bar). ( F-H ) ES cells with the various pnDR-GFP reporters integrated at the Hprt locus were transfected with the indicated Cas9 and gRNA expression vectors to generate single or paired nicks or DSBs (schematically drawn under each bar). ( I ) Schematic representation of the pnDR-GFP0 bp reporter, analogous to (B). ( J ) U2OS cells were transfected with the pnDR-GFP0 bp reporter and the indicated Cas9 and gRNA expression vectors. ( K ) ES cells with pnDR-GFP0 bp integrated at the Hprt locus were transfected with the indicated Cas9 and gRNA expression vectors.

Article Snippet: Cas9 WT , Cas9 D and empty gRNA expression vectors were purchased from Addgene (ID 41815, 41816 and 41824) ( ).

Techniques: Binding Assay, Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

doi: 10.1093/nar/gkw179

Figure Lengend Snippet: Ku suppression of PN HR depends on paired nick offset type and distance. ( A ) Schematic representation of the distinct genetic control of HR triggered by different DNA lesions. The NHEJ component Ku suppresses DSB and PNHR resulting from 5′ overhangs but does not affect nickHR or PNHR resulting from 3′ overhangs. ( B and C ) Ku70 −/− ES-pnDR-GFP-50 bp (B) or Ku70 −/− ES-pnDR-GFP-0 bp (C) cells were transfected with the indicated Cas9 and gRNA expression vectors required to generate single or paired lesions (drawn schematically under each bar) and either the empty (+ Vector) or KU70 (+ KU70) expression vector. ( D–F ) Ku70 −/− ES cells with the various pnDR-GFP reporter variants were transfected with the expression vectors for Cas9 WT (D), Cas9 D (E) or Cas9 H (F), gRNA09enh, gRNA10enh and either the empty (+ Vector) or KU70 (+ KU70) expression vector.

Article Snippet: Cas9 WT , Cas9 D and empty gRNA expression vectors were purchased from Addgene (ID 41815, 41816 and 41824) ( ).

Techniques: Control, Transfection, Expressing, Plasmid Preparation

Journal: Current biology : CB

Article Title: Distinct roles of RZZ and Bub1-KNL1 in mitotic checkpoint signaling and kinetochore expansion

doi: 10.1016/j.cub.2018.10.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: empty gRNA vector , G. Church , Addgene 41824.

Techniques: Plasmid Preparation, Recombinant, Clone Assay, Cotransfection, Software

Journal: Nature biotechnology

Article Title: Treatment of acute myeloid leukemia models by targeting a cell surface RNA-binding protein.

doi: 10.1038/s41587-025-02648-2

Figure Lengend Snippet: Fig. 2 | Commercial and new anti-NPM1 antibodies detect csNPM1 across many human and murine AML models. a, Histograms of live cell flow cytometry of nine human leukemia cell lines stained with an isotype (gray) or the commercially available anti-NPM1 FC8791 (orange) antibody. AF, Alexa Fluor. b, Histograms of flow cytometry of four primary murine leukemias isolated from BM-derived cells. AML-driving mutations are noted; all models are on a Flt3ITD/+ background. The top row shows flow cytometry on the surface, and the bottom shows intracellular staining of cells that were first fixed and permeabilized before adding the anti-NPM1 (B0556) or isotype antibodies. c, Histograms of live cell flow cytometry on K562 cells that were transduced with an over expression plasmid containing no complementary DNA (cDNA) (empty vector) or cDNAs encoding Ty1-tagged NPM1-WT (Ty1-NPM1-WT) or the NPM1c mutant (Ty1- NPM1c). Cells were stained either with anti-NPM1 (B0556, left) or anti-Ty1 (right) antibodies. d, Contour plot of OCI-AML3 cell live staining with the mAb2 (red) or

Article Snippet: Lentiviral vector production and infection For virus production, 293FT cells were transfected with the lentiviral vector (lentiCRISPR-v2) either as an empty vector or containing NPM1 guide RNA together with the packaging plasmids psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259).

Techniques: Flow Cytometry, Staining, Isolation, Derivative Assay, Transduction, Over Expression, Plasmid Preparation, Mutagenesis

Journal: Neuron

Article Title: Mutations in spliceosomal genes PPIL1 and PRP17 cause neurodegenerative pontocerebellar hypoplasia with microcephaly

doi: 10.1016/j.neuron.2020.10.035

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Empty CRISPRi plasmid (PX330-U6–2XBsmBI-gRNA-CBh-dCas9-KRAB-T2a-Puro) was generated on the modified PX330 with 2× BsmBI gRNA cloning sites. dCas9-KRAB-T2a-Puro was amplified from vector pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2aPuro (Addgene #71236) and cloned inside PX330 to replace original WT Cas9. gRNAs targeting PRP17 or scramble gRNA was further cloned between 2XBsmBI sites.

Techniques: Virus, Recombinant, Flow Cytometry, cDNA Synthesis, RNAscope, Multiplex Assay, Control, Knock-Out, Knock-In, Mutagenesis, Plasmid Preparation, Software

Journal: Nature methods

Article Title: CRISPR-assisted detection of RNA-protein interactions in living cells.

doi: 10.1038/s41592-020-0866-0

Figure Lengend Snippet: Fig. 1 | CARPID identifies lncRNA XIST-associated proteins in living cells. a, Scheme of the CARPID workflow. Biotin is represented by red circles marked with ‘B’. b, Volcano plot of XIST-associated proteins identified by CARPID. Significantly enriched proteins are shown as orange dots. Proteins previously confirmed to interact with XIST are shown in the orange font (n = 3 and 9 independent experiments for control and XIST group, respectively). Two previously uncharacterized RBPs of XIST identified and validated in this study—TAF15 and SNF2L—are labeled in blue. n.s., nonsignificant. c, Top, Western blot of TAF15 in input and streptavidin immunoprecipitation samples of control (C) and three XIST gRNA sets (L1, L2 and L3). Bottom, immunoFISH images of XIST and TAF15 in HEK293T cells. The white boxed region on the left is magnified and shown on the right. Three independent experiments were carried out with similar results, and a representative result is shown. d, Validation of XIST–TAF15 interaction using RIP (mean ± s.e.m., n = 3 independent experiments, two-sided paired Student’s t-test). XIST: P1, P2 and P3 represent qPCR primer pairs enriched regions covering corresponding XIST gRNA pairs targeting loci L1/L2/L3, specified in Extended Data Fig. 1a. MALAT1: P1 and P2 represent two different qPCR primer pairs enriched regions in MALAT1 transcript as controls. e, RNA-binding specificity of TAF15 as determined using HTR-SELEX. The gkm-SVM scores were calculated by the gkm-SVM model trained with HTR-SELEX data to predict the potential of TAF15 binding to the corresponding locus in XIST transcript.The blue curve shows the predicted binding affinity of TAF15 along XIST, compared with the average value of 1,000 randomly selected genomic fragments (orange). f, X-linked GFP de-repression under depletion of TAF15 with short hairpin RNAs (shRNA; shTAF15-07 and shTAF15-44) and SNF2L (shSNF2L-29 and shSNF2L-31) in iMEF cells, using SMCHD1 (shSMCHD1) as a positive control (mean ± s.d., n = 3 independent experiments, two-sided unpaired Student’s t-test using NT + 5-aza as a control). NT, non-specific-targeting shRNA. 5-aza, 5-aza-2'-deoxycytidine, a DNA-demethylating agent to derepress gene expression in the inactive X chromosome.

Article Snippet: We generated gRNA sets composed of 2 gRNAs spaced by 30-nucleotide direct repeats to target 2 adjacent loci on the same lncRNA and cloned them into an empty gRNA expressing vector (Addgene no. 109054).

Techniques: Control, Labeling, Western Blot, Immunoprecipitation, Biomarker Discovery, RNA Binding Assay, Binding Assay, shRNA, Positive Control, Gene Expression