sgrnas gene  (Addgene inc)

Journal: Epigenetics & Chromatin

Article Title: SETDB1/ATF7IP regulate the precise genome engineering of HUSH-regulated genes

doi: 10.1186/s13072-026-00661-6

Figure Lengend Snippet: Schematic of the CRISPR-mediated whole genome screen (see also Fig. and Supplemental Figures S2, S3). A The top of the schematic shows the Brunello sgRNA library, which encompasses 76,441 sgRNAs for 19,114 endogenous genes (green rectangle). The sgRNAs (blue rounded rectangle and hairpin) were constitutively expressed using a U6 promoter (blue arrow). In addition, productively-infected cells also constitutively express the puromycin resistance gene ( Puro ; orange rounded rectangle) using an EF1α promoter (orange arrow). The middle part of the schematic shows a transgene cassette that inducibly expresses Cas9. From left to right the cassette contains a tetracycline response element (TRE; blue and yellow hatched rectangle), a normally inactive promoter (black arrow with red X), the Cas9 gene (blue rectangle), a constitutive EF1α promoter (black arrow) driving expression of rtTA (the gene = red rectangle and the expressed protein is a red PacMan™) and the neomycin resistance gene ( Neo ; yellow rectangle). The bottom part of the schematic shows the dual-reporter cassette stably integrated at the AAVS1 locus (black line). Gene expression of the cassette is constitutively driven by a CMV promoter (green arrow). The reporter contains two drug resistance genes, one for BSD (red rectangle) and one for mycophenolic acid ( IMPDH ; red rectangle) which can be co-expressed due to the presence of the ribosome-skipping peptide, P2A (orange rectangle). In the indicated configuration, neither BSD nor IMPDH are expressed due to the presence of engineered tandem stop codons (2X stop signs and green rectangles) in their respective open reading frames (see also Supplemental Figures S2, S3). This panel corresponds to T(-3) > T0 in Fig. . B In the presence of the inducer, Dox (+ Dox, yellow triangle; also referred to as Track A), or Cas9 mRNA (green squiggly line) transfections (see text and Fig. ; also referred to as Track B), Cas9 protein (blue circles) is expressed, which facilitates the functional inactivation of the 19,114 target genes (red X over green rectangle). This panel corresponds to T0 > T3 in Fig. . C After 3 days, Dox was washed out (Track A) or the Cas9 mRNA was given time to decay (Track B). This panel corresponds to T3 > T7 of Fig. . D nCas9 (light blue circles) protein along with a sgRNA targeting the dual stop codon region in the BSD gene was introduced into the cells along with an ssDI donor ODN that encoded a portion of the wild type BSD open reading frame to correct the double stop codon. Over the course of the next 11 days, corrected cells expressing BSD resistance (green rectangle) were selected for. This panel corresponds to T11 > T18 of Fig. . E nCas9 protein was again introduced into the cells, along with a sgRNA targeting the dual stop codon region in the IMPDH gene. This time, an ssDI donor ODN that encoded a portion of the wild type IMPDH open reading frame to correct the double stop codon was utilized. Over the course of the next 15 days, corrected cells expressing IMPDH resistance (green rectangle) were selected for. This panel corresponds to T18 > T33 of Fig.

Article Snippet: For individual gene KOs in HCT116 cells, 1 × 10 6 cells in 10 μL Neon buffer R were combined with 1 μL CleanCap Cas9 mRNA (TriLink) along with 1 μL of 100 mM gene specific sgRNA (Synthego) and electroporated with 1 pulse at 1530 volts for 30 msec in a 10 μL Neon tip.

Techniques: CRISPR, Infection, Expressing, Stable Transfection, Gene Expression, Transfection, Functional Assay

Journal: Epigenetics & Chromatin

Article Title: SETDB1/ATF7IP regulate the precise genome engineering of HUSH-regulated genes

doi: 10.1186/s13072-026-00661-6

Figure Lengend Snippet: Flow diagram of the CRISPR-mediated whole genome KO strategy. From left to right: a Brunello sgRNA library, which encompasses 76,441 sgRNAs for 19,114 genes was infected into HAP1 cells at (arbitrarily) day − 3 [T(-3)]. The cells were treated for 3 days with puromycin ( Puro ) to select for the cells which had productively taken up a sgRNA lentivirus. After 3 days, doxycycline (+ Dox, blue rectangle) induction was started at (T0) on half of the population. At day (T1) the other half of the population was transfected with Cas9 (+ Cas9) mRNA. Henceforth the + Dox-only sample was referred to as the “A Track” and the + Cas9 mRNA sample as the “B track”. At day 7 (T7) a ssDI experiment was performed on both populations of cells (approximately 70 million cells each) to functionally correct an imbedded, but inactive BSD gene (see also Fig. and Supplemental Figures S2, S3). After 48 h (i.e., T9) a blasticidin selection was started for both populations of cells. After an additional 24 h (T10) a sample of cells was saved from both tracks for subsequent bioinformatics analysis. After a week (T18) a second ssDI experiment was performed on both populations of cells (again, approximately 70 million cells each) to functionally correct an imbedded, but inactive IMPDH gene (see also Fig. and Supplemental Figures S2, S3). After 72 h (T21) a mycophenolic acid selection was started. 12 days later (T33) the experiment was terminated for the A and B tracks of cells, which were now resistant to both BSD and mycophenolic acid (BSD + :IMPDH + ). In addition, a sample of cells (T33) was saved from both tracks for subsequent bioinformatics analysis (see also Fig. ).

Article Snippet: For individual gene KOs in HCT116 cells, 1 × 10 6 cells in 10 μL Neon buffer R were combined with 1 μL CleanCap Cas9 mRNA (TriLink) along with 1 μL of 100 mM gene specific sgRNA (Synthego) and electroporated with 1 pulse at 1530 volts for 30 msec in a 10 μL Neon tip.

Techniques: CRISPR, Infection, Transfection, Selection

Journal: Nature Communications

Article Title: Tight junction-high and CDH17-positive cell population is the source of colorectal cancer liver metastases

doi: 10.1038/s41467-025-68169-3

Figure Lengend Snippet: A , B Schematic representation of the strategy used for the vivo intrasplenic metastasis assay with PDO5 in nude mice and photograph of the liver metastases ( A ); and bar chart with the quantification of the number of macroscopic metastases in the different experimental conditions WT, IKKα KO and WT followed by vemurafenib treatment ( B ) (n = 8 (WT), 7 (KO) and 8 (WT+Vemur.) mice examined). C Representative microscopic images of the immunohistochemistry analysis of the activated fibroblast marker SMA and the proliferation marker KI67 in the livers of mice transplanted with PDO5 of the indicated genotypes, and quantification of the data ( n = 6 (WT) and 8 (KO) tumor samples examined). D , E Kaplan-Meier curves of disease-free (DFS) and overall survival (OS) over time for CRC patients included in the metacohort according to CHUK levels using the optimal values as cutoff for patient stratification. Patients at stages II + III ( D ) or all stages ( E ) were included in the analysis. Kaplan-Meier curve estimates are shown with a 95% confidence interval. Statistical tests; B Data are presented as mean ± SD, ordinary one-way ANOVA and Tukey’s multiple comparisons test (n.s. p -value = 0.7671); C Data are presented as box plots showing the median, the 25th–75th percentiles, and the minimum and maximum values, unpaired two-tailed t test (n.s. p -value > 0.05). Source data are provided as a Source Data file.

Article Snippet: The sgRNA against CHUK gene was designed using Benchling and is available at Supplementary Table .

Techniques: Immunohistochemistry, Marker, Clinical Proteomics, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: The rs11067228 risk enhancer is a hub for intra- and inter-chromosomal interactions. (A) Circos plot showing genome-wide interactions indicated by curves extending from the enhancer bait locus. For each curve, cis-interactions are depicted in orange and trans-interactions are depicted in blue. Interactions reproducible in two biological replicates are shown. (B) Comparison of fold-changes in gene expression based on RNA-seq and corresponding 4C-Seq signal counts. Boxed genes indicate overlapping down-regulated genes in enhancer KO relative to WT lines with numbers of genome-wide interactions, as indicated by 4C-Seq (log2fold-change < -1.3; p-adjusted value <0.05). (C) Real-time qPCR validation of transcript levels of potential target genes of the risk enhancer based on analysis in enhancer KO cells. Shown are means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (D-G) 3C-PCR and sequencing confirmation of interaction of the rs11067228-associated enhancer with loci harboring NFASC (D), UGT2B15 (E), TBX3 (F) and SRRM4 (G) genes. Chromatograms confirm respective enhancer and target gene sequences flanking a HindIII linker sequence. M: marker. G-DNA: Genomic DNA, serving as PCR template. 3C: PCR products amplified from the DNA fragments in the 3C library. Forward and reverse PCR primers were designed based on sequences at both enhancer and target gene regions, respectively. (H-O) RT-qPCR and western blot analysis validation of mRNA levels of 4 target genes in WT 22Rv1 cells, 3 enhancer KO lines and enhancer-KO cells subjected to CRISPRa to overexpress indicated target genes individually. Data represents means ± S.E.M. of three independent experiments. *** P < 0.001.

Article Snippet: CRISPRa sgRNAs targeting genes regulated by the rs11067228-related enhancer were designed and cloned into lentiSAMv2 plasmid (75112, Addgene) using the restriction enzyme BsmBI, and packaged and infected as described above.

Techniques: Genome Wide, Comparison, Gene Expression, RNA Sequencing, Biomarker Discovery, Sequencing, Marker, Amplification, Quantitative RT-PCR, Western Blot

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: UGT2B15 and SRRM4 are essential for the development and maintenance of neuroendocrine differentiation of PCa cells. (A, B) Results of soft agar colony formation assays of WT 22Rv1 cells and three lines each of enhancer KO lines, plus enhancer KO cells subjected to CRISPRa to re-express either UGT2B15 (A) or SRRM4 (B) (left). Scale bar =100 μm. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001. (C, D) Transwell assays of cells described in (A, B) (left). Scale bar =100 μm. Cells migrated to lower chambers were stained with 0.1% crystal violet. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001. (E-H) LDH assays in enzalutamide-treated cells corresponding to those described in (A, B). LDH release was assayed after 24 (E, G) and 48 (F, H) hours of treatment. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (I) MTS assays in enzalutamide-treated cells corresponding to those described in (A, B). Data represent means ± S.E.M. of three independent experiments. ***P < 0.001. (J) Western blot showing NE-related protein ( SYP , CHGA and CHGB ) expression in wild-type 22Rv1 cells, as well as three lines each of enhancer-deleted lines plus enhancer-deleted cells re-expressing (by CRISPRa) SRRM4 . (K) Real-time qPCR validation of transcript levels of NE-related genes in cells corresponding to those described in J. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001.

Article Snippet: CRISPRa sgRNAs targeting genes regulated by the rs11067228-related enhancer were designed and cloned into lentiSAMv2 plasmid (75112, Addgene) using the restriction enzyme BsmBI, and packaged and infected as described above.

Techniques: Staining, Western Blot, Expressing, Biomarker Discovery

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: SRRM4 knockdown blocks PCa cell neuroendocrine differentiation. (A-G, R) Validation of target gene knockdown in PCa cells. Expression of indicated target genes, as measured by real-time qPCR and western blot analysis. sgRNAs targeting gene promoter regions were used in CRISPR-interference (CRISPRi) assays. Data represents means ± S.E.M. of three independent experiments. *** P < 0.001. (H, I) Soft agar colony formation assays in WT control 22Rv1 cells and in cells made deficient in indicated target genes using CRISPRi (left). Scale bar =100 μm. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001. (J, K) Transwell assays of cells described in (H, I) (left). Scale bar =100 μm. Cells that had migrated to lower chambers were stained with 0.1% crystal violet. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001. (L-O) LDH assays of enzlutamide-treated cells described in (H, I). Assays were performed after 24 (L, N) and 48 (M, O) hours of treatment. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (P) MTS assays of enzlutamide-treated cells described in (H, I). Data represent means ± S.E.M. of three independent experiments. ***P < 0.001. (Q) Real-time qPCR validation of transcript levels of indicated NE-related genes ( SYP , CHGA and CHGB ) in cells made deficient in SRRM4 using CRISPRi. Data represent means ± SEM of three independent experiments. ***P < 0.001. (R) Western blot showing NE-related protein expression in cells made deficient in SRRM4 using CRISPRi.

Article Snippet: CRISPRa sgRNAs targeting genes regulated by the rs11067228-related enhancer were designed and cloned into lentiSAMv2 plasmid (75112, Addgene) using the restriction enzyme BsmBI, and packaged and infected as described above.

Techniques: Knockdown, Biomarker Discovery, Expressing, Western Blot, CRISPR, Control, Staining

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: SNP rs11067228 is a functional SNP in 22Rv1 cells. (A) Transwell assays in 22Rv1 cells homozygous for the non-risk (G/G, WT) versus risk (A/A, MUT) alleles of rs11067228. Scale bar =100 μm. Cells that had migrated to lower chambers were stained with 0.1% crystal violet. Quantification is at right. Data represent means ± SEM of three independent experiments. **P < 0.01. (B) Soft agar colony formation analysis of WT (G/G) versus MUT (A/A) 22Rv1 cells. Scale bar =100 μm. Quantification is at right. Data represent means ± SEM of three independent experiments. **P < 0.01. (C, D) LDH assays comparing WT (G/G) and MUT (A/A) cells treated with enzalutamide. LDH release was assayed at 24 (C) and 48 (D) hours after treatment. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (E) MTS assays comparing WT (G/G) and MUT (A/A) cells treated with enzalutamide. Data represent means ± S.E.M. of three independent experiments. **P < 0.01. (F) Real-time qPCR validation of transcript levels of indicated NE-related genes in WT (G/G) versus MUT (A/A) cells. Data represent means ± SEM of three independent experiments. ***P < 0.001, **P < 0.01. (G) Heatmap of differentially-expressed genes in two samples of 22Rv1 cells harboring the non-risk (G) allele (WT) and two groups of MUT cells harboring the risk (A) allele, based on RNA-seq (|log2fold change| >1 and p-adjusted value <0.05). (H) KEGG pathway showing biological processes of genes upregulated in MUT relative to WT cells (Top 10). (I) Overlap of genes down-regulated in enhancer KO versus WT lines (p-adjusted value <0.05) (green), with the number of genes up-regulated in lines harboring MUT (A/A) versus WT (G/G) cells (log2fold-change >1; p-adjusted value <0.05) (blue). (J, K) Real-time qPCR and western blot analysis validation of transcript levels of indicated target genes of the risk enhancer in MUT and WT cells. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (L) Real-time qPCR validation of transcript levels of EMT-related genes in WT (G/G), enhancer KO, and MUT (A/A) cells. Data represent means ± SEM of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05. (M-O) Photograph showing 22Rv1 xenografts in nude mice with WT (G/G), enhancer KO, enhancer KO cells subjected to CRISPRa to re-express SRRM4 and MUT (A/A). Tumor volume was measured once a week at indicated time points. Tumor weight was measured when sacrificed (n = 6 per group). Data represent means ± SEM of three independent experiments. ***P < 0.001, **P < 0.01, n.s., not significant. (P) Representative immunohistochemistry staining of indicated 22Rv1 xenografts in (M). Scale bar =100 μm.

Article Snippet: CRISPRa sgRNAs targeting genes regulated by the rs11067228-related enhancer were designed and cloned into lentiSAMv2 plasmid (75112, Addgene) using the restriction enzyme BsmBI, and packaged and infected as described above.

Techniques: Functional Assay, Staining, Biomarker Discovery, RNA Sequencing, Western Blot, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: The castration-resistant prostate cancer-associated SNP rs11067228 facilitates neuroendocrine differentiation through an enhancer-mediated chromatin interaction with SRRM4

doi: 10.7150/ijbs.124731

Figure Lengend Snippet: The transcription factor SOX4 preferentially bind to the rs11067228 risk allele to modulate target gene expression. (A) Schematic showing DNA-protein pull-down assays and mass spectrometry (MS) analysis to identify TFs binding to the rs11067228 risk allele. (B) Overlap in the number of TFs significantly enriched at the risk (A) relative to non-risk (G) allele (orange) with numbers of predicted TF motifs analyzed in the JASPAR database (blue). (C) The sequence of a potential SOX4 binding motif differs in the risk (A) versus non-risk (G) alleles of rs11067228. (Upper) Predicted preferential SOX4 binding site, based on the JASPAR database. (Lower) Shown are actual sequences of non-risk and risk alleles. (D) Quantification of ChIP-qPCR analysis showing SOX4 enrichment at the rs11067228 locus in indicated WT (G/G) versus MUT (A/A) cells. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001, n.s., not significant. (E) Quantification of ChIP-qPCR analysis showing H3K27ac enrichment at the rs11067228 locus in indicated WT (G/G) versus MUT (A/A) cells. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001, n.s., not significant. (F) Transwell assays of MUT 22Rv1 cells and two lines each of SOX4 knockdown lines, plus SOX4 knockdown cells subjected to CRISPRa to re-express SRRM4 . Cells migrated to lower chambers were stained with 0.1% crystal violet. Scale bar =100 μm. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, n.s., not significant. (G) Results of soft agar colony formation assays of cells described in (F) (left). Scale bar =100 μm. Quantification is at right. Data represents means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01. (H, I) LDH assays in enzalutamide-treated cells corresponding to those described in (F). LDH release was assayed after 24 (H) and 48 (I) hours of treatment. Data represent means ± S.E.M. of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05. (J) Real-time qPCR validation of transcript levels of NE-related genes ( SYP , CHGA and CHGB ) in cells described in (F). Data represent means ± S.E.M. of three independent experiments. ***P < 0.001.

Article Snippet: CRISPRa sgRNAs targeting genes regulated by the rs11067228-related enhancer were designed and cloned into lentiSAMv2 plasmid (75112, Addgene) using the restriction enzyme BsmBI, and packaged and infected as described above.

Techniques: Targeted Gene Expression, Mass Spectrometry, Binding Assay, Sequencing, ChIP-qPCR, Knockdown, Staining, Biomarker Discovery