Journal: bioRxiv

Article Title: Optical Pooled Screening for the Discovery of Regulators of the Alternative Lengthening of Telomeres Pathway

doi: 10.1101/2025.02.15.638448

Figure Lengend Snippet: A) OPS experimental workflow. A lentiviral library containing sgRNAs of interest was transduced into target spCas9-expressing cells at a low multiplicity of infection (MOI), followed by selection for a resistance marker to generate a heterogenous pooled cell library. Cells with an integrated lentiviral vector were then fixed and permeabilized. Subsequently, the mRNA containing the sgRNA sequence was retrotranscribed in situ using an LNA-modified oligo. Next, a padlock probe was hybridized around the sgRNA sequence of the cDNA followed by probe extension and ligation. The circularized probe containing the sgRNA sequence was used as a template to perform rolling circle amplification (RCA) and the resulting amplification product is sequenced using sequencing-by-synthesis (SBS) chemistry, a form of in situ sequencing (ISS). B) Representative images of 3 ISS cycles using 4-color SBS chemistry. Scale bar: 20 microns. C) Boxplots showing the number of RCA spots per cell detected in three different cell lines expressing 5 sgRNAs at a time and using conventional OPS protocol. The middle line represents the 50 th percentile. The edges of the box represent the 25 th and 75 th percentile, respectively. The whiskers extend to 1.5 * Inter Quantile Range (IQR). D) Plot showing the inverse relationship between the length of the sgRNA read and genotyping accuracy when using ISS. E) Graph illustrating the direct relationship between spot quality threshold and genotyping accuracy when using ISS. F) Bar plot showing the percentage of lamin A-positive cells when hTERT-RPE-Cas9 cells are transduced with just a control non-targeting sgRNA (sgCTL) or an equimolar mix of 3 LMNA -targeting and 2 scrambled/non-targeting sgRNAs (sgLMNA). Mean± SD. G) Example images of hTERT-RPE-Cas9-sgLMNA cells stained with an anti-lamin A primary antibody and a Alexa488-conjugated secondary antibody together with a segmentation and sgRNA classification mask. Scale bar, 20 microns. H) Density plot showing the distribution of lamin A mean intensity values across hTERT-RPE-Cas9-sgLMNA cells. The proportion of cells with lower lamin A intensity values is larger in cells expressing LMNA -targeting sgRNAs in comparison to cells expressing non-targeting sgRNAs.

Article Snippet: Either the CRISPick [ ] or sgRNA Scorer [ ] web-based software was used to design sgRNA CRISPR knock-out (KO) sequences targeting genes of interest as well as non-targeting controls. sgRNA sequences were individually synthesized (IDT) and independently cloned into lentiviral sgRNA expression vectors, either CROPseq-Guide-Puro (Addgene #86708) or CROPseq-puro-v2 (Addgene #127458), using NEBuilder HiFi DNA Assembly (NEB #E5520S) and BsmBI (NEB #0739S) restriction sites.

Techniques: Expressing, Infection, Selection, Marker, Plasmid Preparation, Sequencing, In Situ, Modification, Ligation, Amplification, Transduction, Control, Staining, Comparison

Journal: bioRxiv

Article Title: Optical Pooled Screening for the Discovery of Regulators of the Alternative Lengthening of Telomeres Pathway

doi: 10.1101/2025.02.15.638448

Figure Lengend Snippet: A) OPS experimental workflow illustrating the steps in the ISS library preparation where the nFISH protocol can be integrated. B) Example nFISH and ISS images when both protocols are performed individually in U2OS-Cas9-sgScramble cells. ISS/RCA spot images are obtained after hybridizing a fluorescent probe against a constant region of the sgRNA-padlock probe construct previously amplified by RCA. Scale bar: 20 microns. C) Representative nFISH and ISS images when the phenotyping protocol is performed after retrotranscription (RT) and post-fixation (Option i) in U2OS-Cas9-sgScramble cells. (Top panel) nFISH images when a PNA-TelG probe is used. (Middle panel) nFISH images when RT primer is removed from reaction. (Bottom panel) ISS images showing the lack of RCA spots when ISS and nFISH are combined using Option i. Scale bar: 20 microns. D) Representative nFISH and ISS images when the phenotyping protocol is performed after RCA reaction (Option ii) and using different telomeric probes in U2OS-Cas9 cells transduced with sgCTL. (Top panel) nFISH images when the PNA-TelG probe is used. (Middle panels) nFISH images when oligo ssDNA TelG or oligo ssDNA TelC are used. (Bottom panel) Example RCA spot images regardless of telomeric probe used for nFISH. Scale bar: 20 microns. E) Bar plot of the difference in mean RCA spots per cell between ISS protocol performed individually or in combination with nFISH using Option ii. Mean± SD. F) Plot showing the difference in telomeric integrated fluorescence intensity between 24 h and 48 h of oligo ssDNA TelC probe hybridization. Mean± SD. G) Bar plot illustrating how increasing the oligo ssDNA TelC probe concentration results in a steady increase in telomeric integrated fluorescence intensity. Mean± SD. H) Example nFISH images showing the increase in telomeric signal after U2OS-Cas9-sgScamble cells are treated with 10 mM of a TLK inhibitor (TLKi) for 6 h. Scale bar: 20 microns. I) Quantification of telomeric integrated fluorescence intensity for non-treated and TLKi-treated U2OS-Cas9-sgScramble cells. Mean± SD.

Article Snippet: Either the CRISPick [ ] or sgRNA Scorer [ ] web-based software was used to design sgRNA CRISPR knock-out (KO) sequences targeting genes of interest as well as non-targeting controls. sgRNA sequences were individually synthesized (IDT) and independently cloned into lentiviral sgRNA expression vectors, either CROPseq-Guide-Puro (Addgene #86708) or CROPseq-puro-v2 (Addgene #127458), using NEBuilder HiFi DNA Assembly (NEB #E5520S) and BsmBI (NEB #0739S) restriction sites.

Techniques: Construct, Amplification, Transduction, Fluorescence, Hybridization, Concentration Assay

Journal: bioRxiv

Article Title: Optical Pooled Screening for the Discovery of Regulators of the Alternative Lengthening of Telomeres Pathway

doi: 10.1101/2025.02.15.638448

Figure Lengend Snippet: A) Quantification of the telomeric integrated fluorescence intensity for U2OS-Cas9 cells transduced with either lentiviral sgCTL alone or with an equimolar mix of 4 lentiviral FANCM -targeting and 2 different scrambled/non-targeting sgRNAs (sgFANCM). Mean± SD . B) Illustration of the lentiviral vector used to evaluate CRISPR efficiency, pXPR-011. This vector expresses the gene EGFP and an EGFP-targeting sgRNA. C) Representative images of uninduced (Dox-) and induced (Dox+) U2OS-iCas9 cells transduced with pXPR-011. Scale bar: 20 microns. D) Bar plot showing the decrease in total EGFP intensity in induced (Dox+) U2OS-iCas9-pXPR-011 cells compared to uninduced (Dox-) cells. E) Graph showing a decrease in the percentage of EGFP-positive U2OS-iCas9-pXPR-011 cells when Cas9 expression is induced with doxycycline compared to uninduced cells. Mean± SD. F) Example nFISH images of uninduced (Dox-) and induced (Dox+) U2OS-iCas9 cells transduced with sgFANCM. Scale bar: 20 microns. G) Plot illustrating the increase in telomeric integrated fluorescence intensity for induced (Dox+) U2OS-iCas9-sgFANCM cells compared to uninduced (Dox-) cells. Mean± SD.

Article Snippet: Either the CRISPick [ ] or sgRNA Scorer [ ] web-based software was used to design sgRNA CRISPR knock-out (KO) sequences targeting genes of interest as well as non-targeting controls. sgRNA sequences were individually synthesized (IDT) and independently cloned into lentiviral sgRNA expression vectors, either CROPseq-Guide-Puro (Addgene #86708) or CROPseq-puro-v2 (Addgene #127458), using NEBuilder HiFi DNA Assembly (NEB #E5520S) and BsmBI (NEB #0739S) restriction sites.

Techniques: Fluorescence, Transduction, Plasmid Preparation, CRISPR, Expressing

Journal: bioRxiv

Article Title: Optical Pooled Screening for the Discovery of Regulators of the Alternative Lengthening of Telomeres Pathway

doi: 10.1101/2025.02.15.638448

Figure Lengend Snippet: A) OPS-nFISH experimental workflow. spCas9 expression was induced in the pooled cell library by treating the cells with 0.05 μg/mL doxycycline for 72h followed by seeding the cells in a 6-well plate using doxycycline-free media. After 48h, cells were fixed and processed for OPS library preparation. Then, telomeric nFISH was performed using the modified oligo ssDNA protocol, followed by SBS reactions to read the sgRNAs expressed in each cell. B) Plot showing the number of sgRNA barcode reads per cell detected during ISS in the pooled cell library. C) Bar plot showing the number of cells expressing each sgRNA in the library. D) Single-cell quantification of telomeric integrated fluorescence intensity associated to each sgRNA. E) Empirical Cumulative Distribution Function (ECDF) plot showing the cumulative distribution of telomeric integrated fluorescence intensity based on the different sgRNAs present in the library.

Article Snippet: Either the CRISPick [ ] or sgRNA Scorer [ ] web-based software was used to design sgRNA CRISPR knock-out (KO) sequences targeting genes of interest as well as non-targeting controls. sgRNA sequences were individually synthesized (IDT) and independently cloned into lentiviral sgRNA expression vectors, either CROPseq-Guide-Puro (Addgene #86708) or CROPseq-puro-v2 (Addgene #127458), using NEBuilder HiFi DNA Assembly (NEB #E5520S) and BsmBI (NEB #0739S) restriction sites.

Techniques: Expressing, Modification, Fluorescence

Journal: bioRxiv

Article Title: Scalable pooled CRISPR screens with single-cell chromatin accessibility profiling

doi: 10.1101/2020.11.20.390971

Figure Lengend Snippet: ( a ) Chromatin remodeling complex subunits and cofactors targeted in the CRISPR library. ( b ) Heatmap of chromatin accessibility Z -scores at transcription factor binding sites (TFBSs) for the different chromatin remodeling complexes targeted in the screen. We converted the fraction of accessible regions for each TFBS into Z -scores (using all cells in the screen). For visualization, we first average over all cells for a particular target gene and then average over all genes in the complex. The histograms ( left ) show the distribution of Z -scores for each complex. The FACT complex is not shown due to a low number of single cells ( n = 75 cells). ( c ) UMAP representation of the genes perturbed in the screen based on the TFBS differential accessibility Z -score profiles. Subunits of the SWI-SNF pBAF complex are labeled with filled circles and gene names. ( d ) The number of transcription factor binding sites with significant differential accessibility for cells that receive a specific genetargeting CRISPR perturbation, as compared to cells that receive a non-targeting (NT) control sgRNA (FDR q ≤ 0.1). SWI/SNF components and co-factors are highlighted in red. ( e ) The percent of ATAC fragments in enhancers and promoters in cells transduced with ARID1A -targeting and NT sgRNAs. Each point is a single cell. K562 enhancer and promoter genome segmentation is from ENCODE (see Methods ). ( f ) CRISPR-targeted chromatin complex genes with significant differential accessibility at enhancers and/or promoters. ( g ) Volcano plots showing significant changes in accessibility at TFBSs in cells transduced with ARID1A (left), SMARCA5 ( middle ) and RCOR1 ( right ) -targeting sgRNAs. Standardized Z -scores are averaged over single cells. Points in red represent TFBSs with a significant change in accessibility (FDR q ≤ 0.1 and | Z -score| > 0.25).

Article Snippet: To generate NIH-3T3 and HEK293FT cells expressing single-guide RNAs (sgRNAs) for the human/mouse experiment, 10 human non-targeting sgRNAs and 10 mouse non-targeting sgRNAs ( Supplementary Table 2 ) were individually synthesized and cloned into the lentiviral transfer vector CROPseq-Guide-Puro (Addgene 86708), which leads to the synthesis of an RNA Pol3 transcript of the Cas9 sgRNA and an RNA Pol2 polyadenylated transcript containing the puromycin resistance gene, a U6 promoter, and the sgRNA.

Techniques: CRISPR, Binding Assay, Labeling, Control, Transduction

Journal: bioRxiv

Article Title: Scalable pooled CRISPR screens with single-cell chromatin accessibility profiling

doi: 10.1101/2020.11.20.390971

Figure Lengend Snippet: ( a ) Heatmap of chromatin accessibility Z -scores at histone and DNA modifications for different CRISPR perturbations ( n = 3 sgRNA per gene). We converted the fraction of accessible regions for each modification into Z -scores (using all cells in the screen). For visualization, we show the average Z -score for all cells receiving a particular sgRNA. ( b ) Distances in the histone and DNA modifications accessibility profiles shown in panel a between sgRNAs targeting different genes and sgRNAs targeting the same gene. The distance metric is 1-(Pearson correlation of the Z -scores). ( c ) Pearson correlation between averaged accessibility Z -scores at histone and DNA modifications of the indicated number of single cells and the average profile of 400 single cells, for cells with either EZH2 -targeting or non-targeting (NT) sgRNAs. ( d ) UMAP representation of chromatin accessibility Z -scores at histone and DNA modifications from single cells receiving either EZH2 or NT sgRNAs. Also shown is the same UMAP representation with single cells colored by TFBS accessibility enrichment scores for CBX2, CBX8, EZH2, POLR2B , and SIRT6 . ( e ) H3K27me3 ChIP-seq coverage at the HOXA-D loci ( top ). Changes in accessibility at the HOXA-D loci in cells transduced with EZH2 -targeting or NT sgRNAs ( bottom ). *** denotes p < 0.001. ( f ) CRISPR-sciATAC fragments mapping to the HOXA locus in cells transduced with EZH2 -targeting or NT sgRNAs ( n = 510 cells per condition). The sum of all ATAC fragments over the entire HOXA locus in cells transduced with EZH2 -targeting and NT sgRNAs is shown on the right. K562 H3K27me3 ChIP-seq coverage is shown at the bottom. ( g ) Gene expression (qPCR) of EZH2, HOXA3, HOXA5, HOXA11A, HOXA13 and HOXD9 for cells transduced with either EZH2 -targeting or NT sgRNAs

Article Snippet: To generate NIH-3T3 and HEK293FT cells expressing single-guide RNAs (sgRNAs) for the human/mouse experiment, 10 human non-targeting sgRNAs and 10 mouse non-targeting sgRNAs ( Supplementary Table 2 ) were individually synthesized and cloned into the lentiviral transfer vector CROPseq-Guide-Puro (Addgene 86708), which leads to the synthesis of an RNA Pol3 transcript of the Cas9 sgRNA and an RNA Pol2 polyadenylated transcript containing the puromycin resistance gene, a U6 promoter, and the sgRNA.

Techniques: CRISPR, Modification, ChIP-sequencing, Transduction, Gene Expression