grna oligo pools  (Twist Bioscience)

Journal: Scientific Data

Article Title: Comprehensive genome-scale CRISPR knockout screening of CHO cells

doi: 10.1038/s41597-025-04438-6

Figure Lengend Snippet: Schematic illustration of a non-viral, genome-scale CRISPR screening platform in Chinese hamster ovary (CHO) cells. ( a ) Plasmid library construction. CHO genome-wide CRISPR guide RNA (gRNA) oligonucleotides were designed and synthesized. Recombinase mediated cassette exchange (RMCE) donor plasmid library was then constructed by cloning gRNA oligos into a backbone plasmid that contained a promoter-less puromycin resistance (PuroR) gene and gRNA scaffold, flanked by attB and mutant attB recombination sites. The plasmid library was verified using next-generation sequencing (NGS) analysis. ( b ) Workflow of cell library and knockout (KO) library development. The CHO-K1 host and recombinant cell-based master cell lines (MCLs) were established through CRISPR/Cas9-mediated site-specific integration (SSI) of the landing pad (LP) plasmid donor, which contained mCherry and hygromycin resistance (HygR) gene flanked by attP and mutant attP recombination sites. The host and recombinant cell libraries were generated through the Bxb1-att recombination system and puromycin selection. Promoter-trapping ensures the expression of a single gRNA per cell. The host and recombinant KO libraries were then generated through transient Cas9 expression and blasticidin semi-selection. The distribution of gRNAs in the pooled cell libraries and KO libraries was confirmed using NGS analysis. The KO libraries were also analyzed after long-term cultivation. ( c ) Functional genomic screens. Phenotypes of interest in the KO library can be enriched through reporter-based fluorescence-activated cell sorting (FACS) or pool selection under selective pressure. Highly productive recombinant cell populations were sorted in this study. The distribution of gRNAs was confirmed using NGS analysis. The screening hits are candidate targets for KO cell engineering.

Article Snippet: The purified gRNA oligo pool was cloned into a plasmid backbone, which was digested with FastDigest Esp3I enzyme (Thermo Fisher Scientific), using Gibson Assembly Master Mix (New England Biolabs, Ipswich, MA, USA).

Techniques: CRISPR, Plasmid Preparation, Genome Wide, Synthesized, Construct, Cloning, Mutagenesis, Next-Generation Sequencing, Knock-Out, Recombinant, Generated, Selection, Expressing, Functional Assay, Fluorescence, FACS

Journal: Scientific Data

Article Title: Comprehensive genome-scale CRISPR knockout screening of CHO cells

doi: 10.1038/s41597-025-04438-6

Figure Lengend Snippet: The development process of the cell and KO libraries using a non-viral genome-scale CRISPR screening platform. Entire profiles of ( a ) viability and ( b ) cell growth (VCD; viable cell density) during the generation of the cell (red solid lines) and KO (blue solid lines) libraries in both CHO-K1 host and recombinant cells. Arrows indicate the transfection of RMCE (red) and Cas9 (blue). Shaded areas denote periods of chemical treatment for cell library enrichment (red, puromycin) and KO library enrichment (blue, blasticidin). The pEGFP-c1 expression vector was used as the transfection control (green lines), while chemically untreated cells served as non-enriched controls (dotted lines). ( c ) The percentage of mCherry-negative populations in the CHO-K1 host (blue line) and recombinant cells (red line) during cell library development with and without puromycin selection. Dotted line indicates the threshold required to maintain 500 × gRNA coverage. The mCherry negativity in enriched populations indicated successful implementation of the genome-scale plasmid library into cells. ( d ) Bar plot showing the transfection efficiency of Cas9 in CHO-K1 host cells and recombinant cells as measured through 2 A peptide-linked reporter expression.

Article Snippet: The purified gRNA oligo pool was cloned into a plasmid backbone, which was digested with FastDigest Esp3I enzyme (Thermo Fisher Scientific), using Gibson Assembly Master Mix (New England Biolabs, Ipswich, MA, USA).

Techniques: CRISPR, Recombinant, Transfection, Expressing, Plasmid Preparation, Control, Selection

Journal: Scientific Data

Article Title: Comprehensive genome-scale CRISPR knockout screening of CHO cells

doi: 10.1038/s41597-025-04438-6

Figure Lengend Snippet: Overview of gRNA library NGS data collection. ( a ) Data collection during cell and KO library generation. The CHO genome-wide gRNA library, containing 111,651 unique gRNAs targeting 21,585 genes, was designed, cloned, and introduced into CHO-K1 host and recombinant cells via RMCE. Cell libraries underwent genome-wide KO (Cas9 short-term library) and prolonged cultivation (Cas9 long-term library) for data collection. Genome-wide gRNA representation in plasmid and cell libraries was validated, and gRNA distribution in the Cas9 short-term and long-term libraries was analyzed. Core and conditional essential genes can be identified as significantly depleted genes, depending on the specific cell lines and culture durations. ( b ) Data collection during positive selection. The highly productive recombinant Cas9 short-term library was enriched using the cold capture secretion assay. A control recombinant cell library underwent the same experimental procedure to exclude false positive hits. Functional genes associated with the phenotype of interest were identified as significantly enriched gRNAs. The generated NGS dataset is indicated by asterisks, with the number of asterisks representing the biological replicates: black for the plasmid library, blue for CHO-K1 host, and red for recombinant cells. In total, 13 NGS datasets were collected. S, short-term; L, long-term; FP, false positive; HP, highly productive; -, significantly depleted gRNAs; and + , significantly enriched gRNAs.

Article Snippet: The purified gRNA oligo pool was cloned into a plasmid backbone, which was digested with FastDigest Esp3I enzyme (Thermo Fisher Scientific), using Gibson Assembly Master Mix (New England Biolabs, Ipswich, MA, USA).

Techniques: Genome Wide, Clone Assay, Recombinant, Plasmid Preparation, Selection, Control, Functional Assay, Generated

Journal: Scientific Data

Article Title: Comprehensive genome-scale CRISPR knockout screening of CHO cells

doi: 10.1038/s41597-025-04438-6

Figure Lengend Snippet: Representation of read count distribution. CHO-K1 host and recombinant cells were transfected with the plasmid library and Bxb1 recombinase to generate a cell library. The cell library pool was transfected with a Cas9 plasmid. After 16 d, Cas9 short-term library cells were generated. After 37 d, a Cas9 long-term library cells were generated. The gRNA sequences in each library cell pool were sequenced using NGS. ( a ) Read count number of each gRNA within the plasmid and host cell libraries. ( b ) Cumulative percentages of sequencing reads in the plasmid, host cell, host Cas9 short-term, and host Cas9 long-term libraries. ( c ) Read count distributions of gRNAs after Cas9 transfection in the host cell, host Cas9 short-term, and host Cas9 long-term libraries. ( d ) The read count number of each gRNA within the plasmid and recombinant cell libraries. ( e ) Cumulative percentages of sequencing reads in the plasmid, recombinant cell, recombinant Cas9 short-term, and recombinant Cas9 long-term libraries. ( f ) Read count distributions of gRNAs after Cas9 transfection in the recombinant cell, recombinant Cas9 short-term, and recombinant Cas9 long-term libraries. In a, b, d, and e, the dashed lines indicate an ideal distribution in the gRNA library. Heat maps showing fold changes (FC) in ( g ) all genes and ( h ) statistically significant genes from MAGeCK α-RRA analysis. The gRNA read counts from the host Cas9 short-term and Cas9 long-term libraries were compared to those from the host cell library. The gRNA read counts from the recombinant Cas9 short-term and Cas9 long-term libraries were compared to those from the recombinant cell library. A gene was considered significant at a P -value threshold of 0.01. FC are represented on a color gradient scale from red to blue. The N/A value was assigned a white color. Cell, cell library after plasmid library transfection; Cas9 short, Cas9 short-term library after 16 d; and Cas9 long, Cas9 long-term library after 37 d.

Article Snippet: The purified gRNA oligo pool was cloned into a plasmid backbone, which was digested with FastDigest Esp3I enzyme (Thermo Fisher Scientific), using Gibson Assembly Master Mix (New England Biolabs, Ipswich, MA, USA).

Techniques: Recombinant, Transfection, Plasmid Preparation, Generated, Sequencing

Journal: bioRxiv

Article Title: Enhancer hubs govern chromatin topology and Th17 identity

doi: 10.64898/2026.04.02.715458

Figure Lengend Snippet: a) Genome browser plot of the Il17a / Il17f locus (70kb window) integrating 500bp resolution region capture Micro-C (RCMC; ICE balanced, normalized by observed/expected), with 3D contacts annotated by dashed line and Il17a-5 enhancer contacts indicated by blue triangles; ATAC-STARR-seq pooled input DNA library coverage track containing DNA fragments from Th0 Th1 Th2 Th17 and Treg ATAC-seq (grey); ATAC-STARR-seq activity score (Log2 fold change CPM) from Th0 (blue), Th1 (orange), Th2 (red), Th17 (yellow) and Treg (green) RNA versus Input DNA; Effect sizes for gRNA in CRISPRi for Il17a and Il17f (grey = tested; red = FDR < 0.05). OCRs are labeled with direction (+/-) and distance (in Kbp) relative to nearest gene. b) Scatter plot comparing sgRNA effect sizes (Log2 fold change high vs low bin) for CRISPRi screens using Il17a and Il17f reporters (green = only Il17f, red = only Il17a, blue = both, grey = non-significant; FDR < 0.05). c) Distribution of elementwise sgRNA effect sizes grouped by top functional OCRs in both Il17a (left) and Il17f (right) CRISPRi screens (lines = tested gRNA per element, blue = FDR < 0.05). Density plot (top) shows distribution of effect sizes for all gRNA. d) Flow cytometry analysis summarizing frequency of IL-17a+ cells or e) geometric MFI of Il17f (HCR-FlowFish) expression from in vitro derived Th17 cells following CRISPRi-mediated perturbation with candidate gRNAs. f) Representative stacked histograms to show distribution of in vitro derived Th17 cell Il17a and Il17f signal (red) relative to non-transduced (grey) following CRISPRi-mediated repression with top candidate single gRNA. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-hoc test versus NTC and sandwich standard error ( d ) or one-sample t-tests with Benjamini-Hochberg correction (e) . Data are shown as mean ± s.e.m. for gRNA-transduced (Thy1.1 + ) relative to non-transduced (Thy1.1-) cell signal; *** p<0.001; ** p<0.0001; * p<0.05.

Article Snippet: The final gRNA row-wise data tables are described in Extended Data Table 4. gRNA oligo pools were ordered from Twist Bioscience.

Techniques: Activity Assay, Labeling, Functional Assay, Flow Cytometry, Expressing, In Vitro, Derivative Assay

Journal: bioRxiv

Article Title: Enhancer hubs govern chromatin topology and Th17 identity

doi: 10.64898/2026.04.02.715458

Figure Lengend Snippet: a) Schematic of the CRISPR-based screening workflow for identifying regulatory elements involved in Th17 differentiation. Naive CD4+ T cells were activated in vitro under Th0 conditions for 24h, followed by transduction with gRNAs targeting open chromatin regions. Cells were then polarized under Th17 conditions for 3 days and prepared for FACS using one of three readouts: i) eGFP expression (i.e Il17a), ii) fixed intracellular staining (i.e RORγt, BATF), or iii) hybridized chain reaction fluorescence in situ hybridization (i.e Il17a Il17f). Cells were finally sorted into high or low expression bins where gRNA abundance was compared. b) Scatter plot comparing sgRNA effect sizes (Log2 fold change hi/lo) between Th17 differentiation noncoding CRISPRi screens with Il17a-eGFP and RORγt readouts (blue = sgRNA significant in both; padj < 0.05). c) Distribution of element-wise effect sizes for gRNA (vertical lines) targeting OCRs in the Il17a- and RORγt-CRISPRi screens (lines = element-targeting gRNA, blue = padj < 0.05). d) Volcano plots depicting sgRNA effect sizes (Log2 fold change) comparing high/low bins for Il17a-eGFP (left) and RORγt(right), with top gRNA labelled (red = padj < 0.05). e ) Mean fluorescence intensity (MFI) of Il17a-eGFP (left) or RORyt (right) from in vitro derived Th17 cells following CRISPRi-mediated repression with individual candidate gRNAs, shown relative to non-targeting control (NTC). Box plots summarize n=5 per targeting gRNA, n=3 for Th0, n=3 for NTC. Statistical analysis was performed using one-way ANOVA with Dunnett’s test versus the NTC and sandwich standard error. Data are shown as mean ± s.e.m. relative to the NTC; * p <0.01; ** p < 0.001

Article Snippet: The final gRNA row-wise data tables are described in Extended Data Table 4. gRNA oligo pools were ordered from Twist Bioscience.

Techniques: CRISPR, In Vitro, Transduction, Expressing, Staining, Fluorescence, In Situ Hybridization, Derivative Assay, Control

Journal: bioRxiv

Article Title: Enhancer hubs govern chromatin topology and Th17 identity

doi: 10.64898/2026.04.02.715458

Figure Lengend Snippet: a) Genome browser view of Rorc and surrounding region (200k bp region) integrating 500bp RCMC contact map (ICE balanced, observed/expected normalization) and 3D interactions annotated by dotted lines; ATAC-STARR pooled Input library (blue), Th17 ATAC-STARR-seq activity score (Log2 CPM (RNA / DNA); yellow); CRISPRi- and CRISPRa-RORγt effect size (red = sgRNA FDR < 0.05; grey = tested); and OCR annotations label the direction (+/-) and distance (in Kbp) from nearest gene. b) Scatter plot comparing sgRNA effect sizes (Log2 fold change RORγt high vs low bins) from CRISPRi and CRISPRa screens (green = CRISPRa only; red = CRISPRi only; blue = both; grey = nonsignificant; FDR < 0.05). c) Element-wise distribution of effect sizes for both CRISPRi (left) and CRISPRa (right) (lines = element-tested gRNA; blue = FDR < 0.05) d) Zoomed in RCMC contact map (500bp resolution) focusing on the proximal RORγt locus (16kb window) with 3D contacts annotated as dotted lines, notable contact enrichments labelled with blue triangles, and corresponding Th17 ATAC-seq coverage track (blue) e) Frequency of IL-17a (blue) or MFI of RORγt (green) relative to non-targeting control (NTC) for in vitro derived Th17 cells following CRISPRi-mediated perturbation with top candidate gRNA from RORγt screening. f) Representative stacked histograms depicting RORγt and IL17a flow cytometry signal for Th17 cells transduced (Thy1+; blue/green) or nontransduced (Thy1-; grey) with candidate gRNAs. Statistical analysis was performed using one-way ANOVA with Dunnett’s test versus the NTC and sandwich standard error. Data are shown as mean ± s.e.m. relative to the NTC; * p <0.001; † p < 0.05.

Article Snippet: The final gRNA row-wise data tables are described in Extended Data Table 4. gRNA oligo pools were ordered from Twist Bioscience.

Techniques: Activity Assay, Control, In Vitro, Derivative Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Enhancer hubs govern chromatin topology and Th17 identity

doi: 10.64898/2026.04.02.715458

Figure Lengend Snippet: STARR-seq signal (Log2 FC) at all ATAC-STARR-seq tested OCRs within Batf , Rorc(t), and Il17a/f loci categorized by CRISPR-screen result (untested = no CRISPR gRNA coverage). b) Waterfall plot of Th17 ATAC-STARR-seq signal (Log2 fold change RNA/DNA) for OCRs with at least 1 significant gRNA in Il17a-CRISPRi (blue circle) or Il17f-CRISPRi (orange circle), c) RORγt-CRISPRi (blue circle) and RORγt-CRISPRa (red circle) or d) Batf-CRISPRi (blue circle) and Batf-CRISPRa (red circle)

Article Snippet: The final gRNA row-wise data tables are described in Extended Data Table 4. gRNA oligo pools were ordered from Twist Bioscience.

Techniques: CRISPR

Journal: bioRxiv

Article Title: Enhancer hubs govern chromatin topology and Th17 identity

doi: 10.64898/2026.04.02.715458

Figure Lengend Snippet: a) Multimodal view of the Batf locus (100k bp window). Top: Region-capture Micro-C (RCMC) contact map (200bp resolution; ICE balanced), with interactions annotated by dotted lines. Tracks display Th17 ATAC-seq coverage by condition (non-targeting control [NTC] = grey; +19kb CRISPRi = red), Th17 ATAC-STARR-seq activity (Log2 CPM RNA / DNA; yellow), and CRISPRi/CRISPRa screen effect sizes (points indicate tested sgRNA, red = FDR < 0.05). Enhancers are annotated by distance (kb) and direction (+/-) relative to the Batf TSS. b) Scatter plot comparing CRISPRi versus CRISPRa effect sizes (Log2 fold change) for all tested sgRNA. Points coloured by significance (FDR < 0.05). c) Distribution of sgRNA effect sizes at selected elements from CRISPRi (left) and CRISPRa (right) screens (blue = significant; grey = tested) d) Comparison of RCMC contact frequency (500bp resolution) at the Batf locus following transduction with Batf +19kb-targeting (top) or NTC (bottom) sgRNAs in dCas9-KRAB Th17 cells. e) Differential contact map showing Log2 fold-change in interaction frequency (Batf +19kb sgRNA / NTC) f) Aggregate Peak Analysis quantifying contact frequency of interactions between the Batf-TSS (P), Batf +19kb (E1) and Batf +43kb (E2) elements in CRISPRi-mediated Batf +19kb perturbed Th17 cells (red) versus NTC (grey). g) Quantitative comparison of transcriptomic changes measured by RNA-seq (Log2 fold-changes relative to control) or h) chromatin accessibility changes by ATAC-seq (Log2 fold-change relative to control) in Batf-/-(BATF-KO) and CRISPRi-mediated Batf +19kb enhancer perturbation (Batf-gRNA) of in vitro derived Th17 cells (RNA Pearson’s r = 0.78; ATAC Pearson’s r = 0.774). i) MFI of BATF (red) or RORyt (green), and frequency of IL-17A+ (blue) from in vitro derived Th17 cells following CRISPRi-mediated repression of candidate OCRs with single gRNA relative to non-targeting control. Box plots summarise n=3 biological replicates j) Representative stacked histograms for BATF (red) IL-17a (blue) and RORγt (green) protein levels in Th17 cells following CRISPRi-mediated repression of Batf +19kb enhancer compared to nontargeting control (grey). Statistical analysis was performed using one-way ANOVA with Dunnett’s test versus the NTC and sandwich standard errors. Data are shown as mean ± s.e.m. relative to the NTC; * p <0.001.

Article Snippet: The final gRNA row-wise data tables are described in Extended Data Table 4. gRNA oligo pools were ordered from Twist Bioscience.

Techniques: Control, Activity Assay, Comparison, Transduction, RNA Sequencing, In Vitro, Derivative Assay