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    Cellecta Inc pooled crisprko libraries brunello
    a , Cloning strategy. The ampicillin resistance gene (AmpR) was removed from the vector pYJA5. sgRNA1–4 and the trimethoprim resistance gene (TmpR) were fused with three distinct PCR amplicons. All elements were Gibson-assembled to form the qgRNA-pYJA5 plasmid, and transformants were selected with trimethoprim. The detailed structure of qgRNA-pYJA5 full plasmid and qgRNA cassette are depicted. LTR, long terminal repeat; Ψ, packaging signal sequence; PB, piggyBac transposon element; PuroR, puromycin resistance element; hU6, mU6, hH1 and h7SK are ubiquitously expressed RNA polymerase-III promoters; sg, sgRNA. F and R arrows: forward and reverse primers used for single-colony PCRs, Sanger and NGS. b , Representative pYJA5 restriction fragments, 3-fragment PCRs and single-colony PCR of ALPA cloning products after transforming into E. coli and trimethoprim selection. Bbs I digestion of pYJA5 yielded an ~1 kb band of the AmpR element and ~7.6 kb band of the linearized vector (left). After PCR with the corresponding sgRNA primers, the three amplicons showed the expected size of 761 bp, 360 bp and 422 bp on agarose gels, respectively (middle). Single-colony PCR with primers flanking the qgRNA cassette of ALPA cloning products in transformed bacteria plate consistently yielded the expected size (2.2 kb, right). c , Percentage of correct, recombined and mutated qgRNA plasmids in 8 independent ALPA cloning experiments with distinct qgRNA sequences (≥22 colonies were tested in each experiment). d , Percentage of correct, recombined and mutated qgRNA plasmids in four ALPA cloning experiments. Each dot represents an independent biological replicate consisting of eight colonies ( n = 24; mean ± s.e.m.). e , Timeline of ALPA cloning in high-throughput format (h, hours; d, days). Created with BioRender.com . f , Gene activation (qRT-PCR) in HEK293 cells 3 days post-transfection with dCas9-VPR and single (sg1–4) or four sgRNA (qg) plasmids. Additional genes are shown in Extended Data Fig. . Dots (here and henceforth): independent experiments (mean ± s.e.m.). g , Gene ablation efficiency by single sgRNAs versus qgRNAs in HEK293 cells via co-transfection with the Cas9 plasmid. 12 single sgRNAs (sg1–12) from the <t>Brunello,</t> GeCKOv2 and TKOv3 libraries were tested; qgRNA plasmids (qg-A,B,C,D) were assembled with the random combination of sg1–4, sg5–8 and sg9–12, and the 4 least effective single sgRNAs among the 12 sgRNAs, respectively. Outcomes were re-plotted for the four least effective single sgRNAs along with the respective qg-D. hNTo, NT control plasmid; cell-surface proteins were stained with fluorescent-conjugated antibodies and analysed via flow cytometry. h , qgRNA plasmids robustly ablated genes inadequately disrupted by single sgRNAs. Single sgRNAs were assembled into qgRNA plasmids and co-transfected with the Cas9 plasmid into HEK293 cells (as in g ). In f and g , P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test.
    Pooled Crisprko Libraries Brunello, supplied by Cellecta Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pooled crisprko libraries brunello/product/Cellecta Inc
    Average 90 stars, based on 1 article reviews
    pooled crisprko libraries brunello - by Bioz Stars, 2026-03
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    1) Product Images from "Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes"

    Article Title: Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes

    Journal: Nature Biomedical Engineering

    doi: 10.1038/s41551-024-01278-4

    a , Cloning strategy. The ampicillin resistance gene (AmpR) was removed from the vector pYJA5. sgRNA1–4 and the trimethoprim resistance gene (TmpR) were fused with three distinct PCR amplicons. All elements were Gibson-assembled to form the qgRNA-pYJA5 plasmid, and transformants were selected with trimethoprim. The detailed structure of qgRNA-pYJA5 full plasmid and qgRNA cassette are depicted. LTR, long terminal repeat; Ψ, packaging signal sequence; PB, piggyBac transposon element; PuroR, puromycin resistance element; hU6, mU6, hH1 and h7SK are ubiquitously expressed RNA polymerase-III promoters; sg, sgRNA. F and R arrows: forward and reverse primers used for single-colony PCRs, Sanger and NGS. b , Representative pYJA5 restriction fragments, 3-fragment PCRs and single-colony PCR of ALPA cloning products after transforming into E. coli and trimethoprim selection. Bbs I digestion of pYJA5 yielded an ~1 kb band of the AmpR element and ~7.6 kb band of the linearized vector (left). After PCR with the corresponding sgRNA primers, the three amplicons showed the expected size of 761 bp, 360 bp and 422 bp on agarose gels, respectively (middle). Single-colony PCR with primers flanking the qgRNA cassette of ALPA cloning products in transformed bacteria plate consistently yielded the expected size (2.2 kb, right). c , Percentage of correct, recombined and mutated qgRNA plasmids in 8 independent ALPA cloning experiments with distinct qgRNA sequences (≥22 colonies were tested in each experiment). d , Percentage of correct, recombined and mutated qgRNA plasmids in four ALPA cloning experiments. Each dot represents an independent biological replicate consisting of eight colonies ( n = 24; mean ± s.e.m.). e , Timeline of ALPA cloning in high-throughput format (h, hours; d, days). Created with BioRender.com . f , Gene activation (qRT-PCR) in HEK293 cells 3 days post-transfection with dCas9-VPR and single (sg1–4) or four sgRNA (qg) plasmids. Additional genes are shown in Extended Data Fig. . Dots (here and henceforth): independent experiments (mean ± s.e.m.). g , Gene ablation efficiency by single sgRNAs versus qgRNAs in HEK293 cells via co-transfection with the Cas9 plasmid. 12 single sgRNAs (sg1–12) from the Brunello, GeCKOv2 and TKOv3 libraries were tested; qgRNA plasmids (qg-A,B,C,D) were assembled with the random combination of sg1–4, sg5–8 and sg9–12, and the 4 least effective single sgRNAs among the 12 sgRNAs, respectively. Outcomes were re-plotted for the four least effective single sgRNAs along with the respective qg-D. hNTo, NT control plasmid; cell-surface proteins were stained with fluorescent-conjugated antibodies and analysed via flow cytometry. h , qgRNA plasmids robustly ablated genes inadequately disrupted by single sgRNAs. Single sgRNAs were assembled into qgRNA plasmids and co-transfected with the Cas9 plasmid into HEK293 cells (as in g ). In f and g , P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test.
    Figure Legend Snippet: a , Cloning strategy. The ampicillin resistance gene (AmpR) was removed from the vector pYJA5. sgRNA1–4 and the trimethoprim resistance gene (TmpR) were fused with three distinct PCR amplicons. All elements were Gibson-assembled to form the qgRNA-pYJA5 plasmid, and transformants were selected with trimethoprim. The detailed structure of qgRNA-pYJA5 full plasmid and qgRNA cassette are depicted. LTR, long terminal repeat; Ψ, packaging signal sequence; PB, piggyBac transposon element; PuroR, puromycin resistance element; hU6, mU6, hH1 and h7SK are ubiquitously expressed RNA polymerase-III promoters; sg, sgRNA. F and R arrows: forward and reverse primers used for single-colony PCRs, Sanger and NGS. b , Representative pYJA5 restriction fragments, 3-fragment PCRs and single-colony PCR of ALPA cloning products after transforming into E. coli and trimethoprim selection. Bbs I digestion of pYJA5 yielded an ~1 kb band of the AmpR element and ~7.6 kb band of the linearized vector (left). After PCR with the corresponding sgRNA primers, the three amplicons showed the expected size of 761 bp, 360 bp and 422 bp on agarose gels, respectively (middle). Single-colony PCR with primers flanking the qgRNA cassette of ALPA cloning products in transformed bacteria plate consistently yielded the expected size (2.2 kb, right). c , Percentage of correct, recombined and mutated qgRNA plasmids in 8 independent ALPA cloning experiments with distinct qgRNA sequences (≥22 colonies were tested in each experiment). d , Percentage of correct, recombined and mutated qgRNA plasmids in four ALPA cloning experiments. Each dot represents an independent biological replicate consisting of eight colonies ( n = 24; mean ± s.e.m.). e , Timeline of ALPA cloning in high-throughput format (h, hours; d, days). Created with BioRender.com . f , Gene activation (qRT-PCR) in HEK293 cells 3 days post-transfection with dCas9-VPR and single (sg1–4) or four sgRNA (qg) plasmids. Additional genes are shown in Extended Data Fig. . Dots (here and henceforth): independent experiments (mean ± s.e.m.). g , Gene ablation efficiency by single sgRNAs versus qgRNAs in HEK293 cells via co-transfection with the Cas9 plasmid. 12 single sgRNAs (sg1–12) from the Brunello, GeCKOv2 and TKOv3 libraries were tested; qgRNA plasmids (qg-A,B,C,D) were assembled with the random combination of sg1–4, sg5–8 and sg9–12, and the 4 least effective single sgRNAs among the 12 sgRNAs, respectively. Outcomes were re-plotted for the four least effective single sgRNAs along with the respective qg-D. hNTo, NT control plasmid; cell-surface proteins were stained with fluorescent-conjugated antibodies and analysed via flow cytometry. h , qgRNA plasmids robustly ablated genes inadequately disrupted by single sgRNAs. Single sgRNAs were assembled into qgRNA plasmids and co-transfected with the Cas9 plasmid into HEK293 cells (as in g ). In f and g , P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test.

    Techniques Used: Cloning, Plasmid Preparation, Sequencing, Selection, Transformation Assay, Bacteria, High Throughput Screening Assay, Activation Assay, Quantitative RT-PCR, Transfection, Cotransfection, Control, Staining, Flow Cytometry

    a , H4-Cas9-GFP-SQSTM1 cells were transduced with genome-wide pooled lentiviral sgRNA ablation libraries (T.spiezzo and Brunello) and selected with puromycin. Cells in the uppermost and lowermost fluorescence quartile were collected by FACS and sgRNAs were quantified by sequencing genomic DNA. Created with BioRender.com . b , c , Average log 2 FC in GFP high and GFP low samples from T.spiezzo versus Brunello ( b ) and T.spiezzo versus Cellecta ( c ). Autophagy-relevant genes (autophagy, GO:0006914 ) are highlighted in red. d , Autophagy genes enriched in GFP high cells from the T.spiezzo, Brunello and Cellecta screens. The box plot represents the interquartile range. e , Heatmap showing the highest and lowest mean log 2 FC (GFP high versus GFP low ) of genes identified among the top 100 genes in the T.spiezzo, Brunello and Cellecta screens. f , Quantification of log 2 FC of cell count in GFP high versus GFP low populations transduced with T.spiezzo qgRNA lentivirus against NT control or each of the 16 genes selected for validation. The boundaries for GFP high and GFP low cell populations were set according to the NT condition. Dots (here and henceforth): independent experiments, mean ± s.e.m. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. g , An example of GFP-SQSTM1 puncta in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against NT or HNRNPM (see Extended Data Fig. for all genes tested). Dashed lines: cell contours according to the cytosolic GFP signal. h , Percentage of cells with GFP-SQSTM1 puncta (purple) and puncta area (black) in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against 16 genes selected for validation. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test, *** P < 0.001. i , Immunoblotting of LC3-II from H4-Cas9 cells transduced with T.spiezzo qgRNA lentivirus against 10 possible autophagy modulators in the absence (−) or presence (+) of ChQ (100 µM, 6 h). GAPDH: loading control. Two biological repeats were assessed for each condition. j , Representative micrographs of YFP-LC3 in H4-Cas9-cells transduced with T.spiezzo qgRNA lentivirus against NT or each of the 5 genes selected for further validation. H4-Cas9 cells were cultured and treated as described in a and transduced with YFP-LC3 lentiviruses 48–60 h before examining YFP-LC3 puncta. k , Quantification of puncta area of YFP-LC3 of cells and conditions described in j . N = 3 biological repeats. P values were determined by a one-tailed Mann–Whitney test.
    Figure Legend Snippet: a , H4-Cas9-GFP-SQSTM1 cells were transduced with genome-wide pooled lentiviral sgRNA ablation libraries (T.spiezzo and Brunello) and selected with puromycin. Cells in the uppermost and lowermost fluorescence quartile were collected by FACS and sgRNAs were quantified by sequencing genomic DNA. Created with BioRender.com . b , c , Average log 2 FC in GFP high and GFP low samples from T.spiezzo versus Brunello ( b ) and T.spiezzo versus Cellecta ( c ). Autophagy-relevant genes (autophagy, GO:0006914 ) are highlighted in red. d , Autophagy genes enriched in GFP high cells from the T.spiezzo, Brunello and Cellecta screens. The box plot represents the interquartile range. e , Heatmap showing the highest and lowest mean log 2 FC (GFP high versus GFP low ) of genes identified among the top 100 genes in the T.spiezzo, Brunello and Cellecta screens. f , Quantification of log 2 FC of cell count in GFP high versus GFP low populations transduced with T.spiezzo qgRNA lentivirus against NT control or each of the 16 genes selected for validation. The boundaries for GFP high and GFP low cell populations were set according to the NT condition. Dots (here and henceforth): independent experiments, mean ± s.e.m. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. g , An example of GFP-SQSTM1 puncta in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against NT or HNRNPM (see Extended Data Fig. for all genes tested). Dashed lines: cell contours according to the cytosolic GFP signal. h , Percentage of cells with GFP-SQSTM1 puncta (purple) and puncta area (black) in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against 16 genes selected for validation. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test, *** P < 0.001. i , Immunoblotting of LC3-II from H4-Cas9 cells transduced with T.spiezzo qgRNA lentivirus against 10 possible autophagy modulators in the absence (−) or presence (+) of ChQ (100 µM, 6 h). GAPDH: loading control. Two biological repeats were assessed for each condition. j , Representative micrographs of YFP-LC3 in H4-Cas9-cells transduced with T.spiezzo qgRNA lentivirus against NT or each of the 5 genes selected for further validation. H4-Cas9 cells were cultured and treated as described in a and transduced with YFP-LC3 lentiviruses 48–60 h before examining YFP-LC3 puncta. k , Quantification of puncta area of YFP-LC3 of cells and conditions described in j . N = 3 biological repeats. P values were determined by a one-tailed Mann–Whitney test.

    Techniques Used: Transduction, Genome Wide, Fluorescence, Sequencing, Cell Counting, Control, Biomarker Discovery, Western Blot, Cell Culture, One-tailed Test, MANN-WHITNEY

    a , Histogram showing the gating strategy to isolate GFP high and GFP low (upper and lower quartile of GFP fluorescence, respectively) cell populations. b , Percentages of sequencing reads in GFP high , GFP low , and unsorted samples from the T.spiezzo pooled screen that correctly aligned to sgRNA2 (mapped reads 1) or sgRNA3 (mapped reads 2), that did not align to sgRNA2 (unmapped reads 1) or sgRNA3 (unmapped reads 2) and those that aligned and had the correct linkage between sgRNA2 and sgRNA3 (mapped and linked). c - e , Overrepresentation analysis of the top 200 genes enriched in GFP high cell populations from the T.spiezzo ( c ), Brunello ( d ), and Cellecta ( e ) screens. Gene counts and adjusted p-value are represented in each figure. The 10 most significant GO biological processes are shown. f-m , Autophagy-related gene sets including autophagosome assembly ( GO:0000045 , n=174, f ), autophagosome membrane ( GO:0000421 , n=129, g ), autophagy of mitochondrion ( GO:0000422 , n=109, h ), autophagosome ( GO:0005776 , n=198, i ), regulation of autophagy ( GO:0010506 , n=209, j ), positive regulation of autophagy ( GO:0010508 , n=196, k ), macroautophagy ( GO:0016236 , n=180, l ), and lysosomal microautophagy ( GO:0016237 , n=6, m ) using absolute log 2 fold changes in GFP high cell populations from the T.spiezzo, Brunello, and Cellecta screens. The p value was determined by two-way ANOVA. The box plot represents the interquartile range. n , An example of flow cytometry histograms of GFP-SQSTM1 intensity in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against each of the 16 genes selected for validation or a non-targeting control (NT) lentivirus. N = 3 biological repeats. o , An example of GFP-SQSTM1 puncta in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against each of the 16 genes selected for validation or NT controls. N = 3 biological repeats. Cells were demarcated by dashed lines according to the cytosolic GFP signal. p , Quantification of LC3II levels of cells and conditions described in Fig. . All values were normalized to the mean of the two NT repeats (- ChQ) on the same blot. Both the LC3II and normalized LC3II (LC3II/GAPDH) levels were shown to determine whether consistent changes were observed for the two biological repeats of a defined gene to determine promising candidates for further validation.
    Figure Legend Snippet: a , Histogram showing the gating strategy to isolate GFP high and GFP low (upper and lower quartile of GFP fluorescence, respectively) cell populations. b , Percentages of sequencing reads in GFP high , GFP low , and unsorted samples from the T.spiezzo pooled screen that correctly aligned to sgRNA2 (mapped reads 1) or sgRNA3 (mapped reads 2), that did not align to sgRNA2 (unmapped reads 1) or sgRNA3 (unmapped reads 2) and those that aligned and had the correct linkage between sgRNA2 and sgRNA3 (mapped and linked). c - e , Overrepresentation analysis of the top 200 genes enriched in GFP high cell populations from the T.spiezzo ( c ), Brunello ( d ), and Cellecta ( e ) screens. Gene counts and adjusted p-value are represented in each figure. The 10 most significant GO biological processes are shown. f-m , Autophagy-related gene sets including autophagosome assembly ( GO:0000045 , n=174, f ), autophagosome membrane ( GO:0000421 , n=129, g ), autophagy of mitochondrion ( GO:0000422 , n=109, h ), autophagosome ( GO:0005776 , n=198, i ), regulation of autophagy ( GO:0010506 , n=209, j ), positive regulation of autophagy ( GO:0010508 , n=196, k ), macroautophagy ( GO:0016236 , n=180, l ), and lysosomal microautophagy ( GO:0016237 , n=6, m ) using absolute log 2 fold changes in GFP high cell populations from the T.spiezzo, Brunello, and Cellecta screens. The p value was determined by two-way ANOVA. The box plot represents the interquartile range. n , An example of flow cytometry histograms of GFP-SQSTM1 intensity in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against each of the 16 genes selected for validation or a non-targeting control (NT) lentivirus. N = 3 biological repeats. o , An example of GFP-SQSTM1 puncta in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against each of the 16 genes selected for validation or NT controls. N = 3 biological repeats. Cells were demarcated by dashed lines according to the cytosolic GFP signal. p , Quantification of LC3II levels of cells and conditions described in Fig. . All values were normalized to the mean of the two NT repeats (- ChQ) on the same blot. Both the LC3II and normalized LC3II (LC3II/GAPDH) levels were shown to determine whether consistent changes were observed for the two biological repeats of a defined gene to determine promising candidates for further validation.

    Techniques Used: Fluorescence, Sequencing, Membrane, Flow Cytometry, Transduction, Biomarker Discovery, Control



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    pooled crisprko libraries brunello  (Cellecta Inc)
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    Cellecta Inc pooled crisprko libraries brunello
    a , Cloning strategy. The ampicillin resistance gene (AmpR) was removed from the vector pYJA5. sgRNA1–4 and the trimethoprim resistance gene (TmpR) were fused with three distinct PCR amplicons. All elements were Gibson-assembled to form the qgRNA-pYJA5 plasmid, and transformants were selected with trimethoprim. The detailed structure of qgRNA-pYJA5 full plasmid and qgRNA cassette are depicted. LTR, long terminal repeat; Ψ, packaging signal sequence; PB, piggyBac transposon element; PuroR, puromycin resistance element; hU6, mU6, hH1 and h7SK are ubiquitously expressed RNA polymerase-III promoters; sg, sgRNA. F and R arrows: forward and reverse primers used for single-colony PCRs, Sanger and NGS. b , Representative pYJA5 restriction fragments, 3-fragment PCRs and single-colony PCR of ALPA cloning products after transforming into E. coli and trimethoprim selection. Bbs I digestion of pYJA5 yielded an ~1 kb band of the AmpR element and ~7.6 kb band of the linearized vector (left). After PCR with the corresponding sgRNA primers, the three amplicons showed the expected size of 761 bp, 360 bp and 422 bp on agarose gels, respectively (middle). Single-colony PCR with primers flanking the qgRNA cassette of ALPA cloning products in transformed bacteria plate consistently yielded the expected size (2.2 kb, right). c , Percentage of correct, recombined and mutated qgRNA plasmids in 8 independent ALPA cloning experiments with distinct qgRNA sequences (≥22 colonies were tested in each experiment). d , Percentage of correct, recombined and mutated qgRNA plasmids in four ALPA cloning experiments. Each dot represents an independent biological replicate consisting of eight colonies ( n = 24; mean ± s.e.m.). e , Timeline of ALPA cloning in high-throughput format (h, hours; d, days). Created with BioRender.com . f , Gene activation (qRT-PCR) in HEK293 cells 3 days post-transfection with dCas9-VPR and single (sg1–4) or four sgRNA (qg) plasmids. Additional genes are shown in Extended Data Fig. . Dots (here and henceforth): independent experiments (mean ± s.e.m.). g , Gene ablation efficiency by single sgRNAs versus qgRNAs in HEK293 cells via co-transfection with the Cas9 plasmid. 12 single sgRNAs (sg1–12) from the <t>Brunello,</t> GeCKOv2 and TKOv3 libraries were tested; qgRNA plasmids (qg-A,B,C,D) were assembled with the random combination of sg1–4, sg5–8 and sg9–12, and the 4 least effective single sgRNAs among the 12 sgRNAs, respectively. Outcomes were re-plotted for the four least effective single sgRNAs along with the respective qg-D. hNTo, NT control plasmid; cell-surface proteins were stained with fluorescent-conjugated antibodies and analysed via flow cytometry. h , qgRNA plasmids robustly ablated genes inadequately disrupted by single sgRNAs. Single sgRNAs were assembled into qgRNA plasmids and co-transfected with the Cas9 plasmid into HEK293 cells (as in g ). In f and g , P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test.
    Pooled Crisprko Libraries Brunello, supplied by Cellecta Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pooled crisprko libraries brunello/product/Cellecta Inc
    Average 90 stars, based on 1 article reviews
    pooled crisprko libraries brunello - by Bioz Stars, 2026-03
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    a , Cloning strategy. The ampicillin resistance gene (AmpR) was removed from the vector pYJA5. sgRNA1–4 and the trimethoprim resistance gene (TmpR) were fused with three distinct PCR amplicons. All elements were Gibson-assembled to form the qgRNA-pYJA5 plasmid, and transformants were selected with trimethoprim. The detailed structure of qgRNA-pYJA5 full plasmid and qgRNA cassette are depicted. LTR, long terminal repeat; Ψ, packaging signal sequence; PB, piggyBac transposon element; PuroR, puromycin resistance element; hU6, mU6, hH1 and h7SK are ubiquitously expressed RNA polymerase-III promoters; sg, sgRNA. F and R arrows: forward and reverse primers used for single-colony PCRs, Sanger and NGS. b , Representative pYJA5 restriction fragments, 3-fragment PCRs and single-colony PCR of ALPA cloning products after transforming into E. coli and trimethoprim selection. Bbs I digestion of pYJA5 yielded an ~1 kb band of the AmpR element and ~7.6 kb band of the linearized vector (left). After PCR with the corresponding sgRNA primers, the three amplicons showed the expected size of 761 bp, 360 bp and 422 bp on agarose gels, respectively (middle). Single-colony PCR with primers flanking the qgRNA cassette of ALPA cloning products in transformed bacteria plate consistently yielded the expected size (2.2 kb, right). c , Percentage of correct, recombined and mutated qgRNA plasmids in 8 independent ALPA cloning experiments with distinct qgRNA sequences (≥22 colonies were tested in each experiment). d , Percentage of correct, recombined and mutated qgRNA plasmids in four ALPA cloning experiments. Each dot represents an independent biological replicate consisting of eight colonies ( n = 24; mean ± s.e.m.). e , Timeline of ALPA cloning in high-throughput format (h, hours; d, days). Created with BioRender.com . f , Gene activation (qRT-PCR) in HEK293 cells 3 days post-transfection with dCas9-VPR and single (sg1–4) or four sgRNA (qg) plasmids. Additional genes are shown in Extended Data Fig. . Dots (here and henceforth): independent experiments (mean ± s.e.m.). g , Gene ablation efficiency by single sgRNAs versus qgRNAs in HEK293 cells via co-transfection with the Cas9 plasmid. 12 single sgRNAs (sg1–12) from the Brunello, GeCKOv2 and TKOv3 libraries were tested; qgRNA plasmids (qg-A,B,C,D) were assembled with the random combination of sg1–4, sg5–8 and sg9–12, and the 4 least effective single sgRNAs among the 12 sgRNAs, respectively. Outcomes were re-plotted for the four least effective single sgRNAs along with the respective qg-D. hNTo, NT control plasmid; cell-surface proteins were stained with fluorescent-conjugated antibodies and analysed via flow cytometry. h , qgRNA plasmids robustly ablated genes inadequately disrupted by single sgRNAs. Single sgRNAs were assembled into qgRNA plasmids and co-transfected with the Cas9 plasmid into HEK293 cells (as in g ). In f and g , P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test.

    Journal: Nature Biomedical Engineering

    Article Title: Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes

    doi: 10.1038/s41551-024-01278-4

    Figure Lengend Snippet: a , Cloning strategy. The ampicillin resistance gene (AmpR) was removed from the vector pYJA5. sgRNA1–4 and the trimethoprim resistance gene (TmpR) were fused with three distinct PCR amplicons. All elements were Gibson-assembled to form the qgRNA-pYJA5 plasmid, and transformants were selected with trimethoprim. The detailed structure of qgRNA-pYJA5 full plasmid and qgRNA cassette are depicted. LTR, long terminal repeat; Ψ, packaging signal sequence; PB, piggyBac transposon element; PuroR, puromycin resistance element; hU6, mU6, hH1 and h7SK are ubiquitously expressed RNA polymerase-III promoters; sg, sgRNA. F and R arrows: forward and reverse primers used for single-colony PCRs, Sanger and NGS. b , Representative pYJA5 restriction fragments, 3-fragment PCRs and single-colony PCR of ALPA cloning products after transforming into E. coli and trimethoprim selection. Bbs I digestion of pYJA5 yielded an ~1 kb band of the AmpR element and ~7.6 kb band of the linearized vector (left). After PCR with the corresponding sgRNA primers, the three amplicons showed the expected size of 761 bp, 360 bp and 422 bp on agarose gels, respectively (middle). Single-colony PCR with primers flanking the qgRNA cassette of ALPA cloning products in transformed bacteria plate consistently yielded the expected size (2.2 kb, right). c , Percentage of correct, recombined and mutated qgRNA plasmids in 8 independent ALPA cloning experiments with distinct qgRNA sequences (≥22 colonies were tested in each experiment). d , Percentage of correct, recombined and mutated qgRNA plasmids in four ALPA cloning experiments. Each dot represents an independent biological replicate consisting of eight colonies ( n = 24; mean ± s.e.m.). e , Timeline of ALPA cloning in high-throughput format (h, hours; d, days). Created with BioRender.com . f , Gene activation (qRT-PCR) in HEK293 cells 3 days post-transfection with dCas9-VPR and single (sg1–4) or four sgRNA (qg) plasmids. Additional genes are shown in Extended Data Fig. . Dots (here and henceforth): independent experiments (mean ± s.e.m.). g , Gene ablation efficiency by single sgRNAs versus qgRNAs in HEK293 cells via co-transfection with the Cas9 plasmid. 12 single sgRNAs (sg1–12) from the Brunello, GeCKOv2 and TKOv3 libraries were tested; qgRNA plasmids (qg-A,B,C,D) were assembled with the random combination of sg1–4, sg5–8 and sg9–12, and the 4 least effective single sgRNAs among the 12 sgRNAs, respectively. Outcomes were re-plotted for the four least effective single sgRNAs along with the respective qg-D. hNTo, NT control plasmid; cell-surface proteins were stained with fluorescent-conjugated antibodies and analysed via flow cytometry. h , qgRNA plasmids robustly ablated genes inadequately disrupted by single sgRNAs. Single sgRNAs were assembled into qgRNA plasmids and co-transfected with the Cas9 plasmid into HEK293 cells (as in g ). In f and g , P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test.

    Article Snippet: The accumulation of GFP-tagged SQSTM1 provides a reliable proxy of autophagic activity, motivating us to compare the sensitivity and specificity of a pooled T.spiezzo version with the two pooled CRISPRko libraries, Brunello and Cellecta.

    Techniques: Cloning, Plasmid Preparation, Sequencing, Selection, Transformation Assay, Bacteria, High Throughput Screening Assay, Activation Assay, Quantitative RT-PCR, Transfection, Cotransfection, Control, Staining, Flow Cytometry

    a , H4-Cas9-GFP-SQSTM1 cells were transduced with genome-wide pooled lentiviral sgRNA ablation libraries (T.spiezzo and Brunello) and selected with puromycin. Cells in the uppermost and lowermost fluorescence quartile were collected by FACS and sgRNAs were quantified by sequencing genomic DNA. Created with BioRender.com . b , c , Average log 2 FC in GFP high and GFP low samples from T.spiezzo versus Brunello ( b ) and T.spiezzo versus Cellecta ( c ). Autophagy-relevant genes (autophagy, GO:0006914 ) are highlighted in red. d , Autophagy genes enriched in GFP high cells from the T.spiezzo, Brunello and Cellecta screens. The box plot represents the interquartile range. e , Heatmap showing the highest and lowest mean log 2 FC (GFP high versus GFP low ) of genes identified among the top 100 genes in the T.spiezzo, Brunello and Cellecta screens. f , Quantification of log 2 FC of cell count in GFP high versus GFP low populations transduced with T.spiezzo qgRNA lentivirus against NT control or each of the 16 genes selected for validation. The boundaries for GFP high and GFP low cell populations were set according to the NT condition. Dots (here and henceforth): independent experiments, mean ± s.e.m. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. g , An example of GFP-SQSTM1 puncta in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against NT or HNRNPM (see Extended Data Fig. for all genes tested). Dashed lines: cell contours according to the cytosolic GFP signal. h , Percentage of cells with GFP-SQSTM1 puncta (purple) and puncta area (black) in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against 16 genes selected for validation. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test, *** P < 0.001. i , Immunoblotting of LC3-II from H4-Cas9 cells transduced with T.spiezzo qgRNA lentivirus against 10 possible autophagy modulators in the absence (−) or presence (+) of ChQ (100 µM, 6 h). GAPDH: loading control. Two biological repeats were assessed for each condition. j , Representative micrographs of YFP-LC3 in H4-Cas9-cells transduced with T.spiezzo qgRNA lentivirus against NT or each of the 5 genes selected for further validation. H4-Cas9 cells were cultured and treated as described in a and transduced with YFP-LC3 lentiviruses 48–60 h before examining YFP-LC3 puncta. k , Quantification of puncta area of YFP-LC3 of cells and conditions described in j . N = 3 biological repeats. P values were determined by a one-tailed Mann–Whitney test.

    Journal: Nature Biomedical Engineering

    Article Title: Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes

    doi: 10.1038/s41551-024-01278-4

    Figure Lengend Snippet: a , H4-Cas9-GFP-SQSTM1 cells were transduced with genome-wide pooled lentiviral sgRNA ablation libraries (T.spiezzo and Brunello) and selected with puromycin. Cells in the uppermost and lowermost fluorescence quartile were collected by FACS and sgRNAs were quantified by sequencing genomic DNA. Created with BioRender.com . b , c , Average log 2 FC in GFP high and GFP low samples from T.spiezzo versus Brunello ( b ) and T.spiezzo versus Cellecta ( c ). Autophagy-relevant genes (autophagy, GO:0006914 ) are highlighted in red. d , Autophagy genes enriched in GFP high cells from the T.spiezzo, Brunello and Cellecta screens. The box plot represents the interquartile range. e , Heatmap showing the highest and lowest mean log 2 FC (GFP high versus GFP low ) of genes identified among the top 100 genes in the T.spiezzo, Brunello and Cellecta screens. f , Quantification of log 2 FC of cell count in GFP high versus GFP low populations transduced with T.spiezzo qgRNA lentivirus against NT control or each of the 16 genes selected for validation. The boundaries for GFP high and GFP low cell populations were set according to the NT condition. Dots (here and henceforth): independent experiments, mean ± s.e.m. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. g , An example of GFP-SQSTM1 puncta in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against NT or HNRNPM (see Extended Data Fig. for all genes tested). Dashed lines: cell contours according to the cytosolic GFP signal. h , Percentage of cells with GFP-SQSTM1 puncta (purple) and puncta area (black) in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against 16 genes selected for validation. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test, *** P < 0.001. i , Immunoblotting of LC3-II from H4-Cas9 cells transduced with T.spiezzo qgRNA lentivirus against 10 possible autophagy modulators in the absence (−) or presence (+) of ChQ (100 µM, 6 h). GAPDH: loading control. Two biological repeats were assessed for each condition. j , Representative micrographs of YFP-LC3 in H4-Cas9-cells transduced with T.spiezzo qgRNA lentivirus against NT or each of the 5 genes selected for further validation. H4-Cas9 cells were cultured and treated as described in a and transduced with YFP-LC3 lentiviruses 48–60 h before examining YFP-LC3 puncta. k , Quantification of puncta area of YFP-LC3 of cells and conditions described in j . N = 3 biological repeats. P values were determined by a one-tailed Mann–Whitney test.

    Article Snippet: The accumulation of GFP-tagged SQSTM1 provides a reliable proxy of autophagic activity, motivating us to compare the sensitivity and specificity of a pooled T.spiezzo version with the two pooled CRISPRko libraries, Brunello and Cellecta.

    Techniques: Transduction, Genome Wide, Fluorescence, Sequencing, Cell Counting, Control, Biomarker Discovery, Western Blot, Cell Culture, One-tailed Test, MANN-WHITNEY

    a , Histogram showing the gating strategy to isolate GFP high and GFP low (upper and lower quartile of GFP fluorescence, respectively) cell populations. b , Percentages of sequencing reads in GFP high , GFP low , and unsorted samples from the T.spiezzo pooled screen that correctly aligned to sgRNA2 (mapped reads 1) or sgRNA3 (mapped reads 2), that did not align to sgRNA2 (unmapped reads 1) or sgRNA3 (unmapped reads 2) and those that aligned and had the correct linkage between sgRNA2 and sgRNA3 (mapped and linked). c - e , Overrepresentation analysis of the top 200 genes enriched in GFP high cell populations from the T.spiezzo ( c ), Brunello ( d ), and Cellecta ( e ) screens. Gene counts and adjusted p-value are represented in each figure. The 10 most significant GO biological processes are shown. f-m , Autophagy-related gene sets including autophagosome assembly ( GO:0000045 , n=174, f ), autophagosome membrane ( GO:0000421 , n=129, g ), autophagy of mitochondrion ( GO:0000422 , n=109, h ), autophagosome ( GO:0005776 , n=198, i ), regulation of autophagy ( GO:0010506 , n=209, j ), positive regulation of autophagy ( GO:0010508 , n=196, k ), macroautophagy ( GO:0016236 , n=180, l ), and lysosomal microautophagy ( GO:0016237 , n=6, m ) using absolute log 2 fold changes in GFP high cell populations from the T.spiezzo, Brunello, and Cellecta screens. The p value was determined by two-way ANOVA. The box plot represents the interquartile range. n , An example of flow cytometry histograms of GFP-SQSTM1 intensity in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against each of the 16 genes selected for validation or a non-targeting control (NT) lentivirus. N = 3 biological repeats. o , An example of GFP-SQSTM1 puncta in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against each of the 16 genes selected for validation or NT controls. N = 3 biological repeats. Cells were demarcated by dashed lines according to the cytosolic GFP signal. p , Quantification of LC3II levels of cells and conditions described in Fig. . All values were normalized to the mean of the two NT repeats (- ChQ) on the same blot. Both the LC3II and normalized LC3II (LC3II/GAPDH) levels were shown to determine whether consistent changes were observed for the two biological repeats of a defined gene to determine promising candidates for further validation.

    Journal: Nature Biomedical Engineering

    Article Title: Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes

    doi: 10.1038/s41551-024-01278-4

    Figure Lengend Snippet: a , Histogram showing the gating strategy to isolate GFP high and GFP low (upper and lower quartile of GFP fluorescence, respectively) cell populations. b , Percentages of sequencing reads in GFP high , GFP low , and unsorted samples from the T.spiezzo pooled screen that correctly aligned to sgRNA2 (mapped reads 1) or sgRNA3 (mapped reads 2), that did not align to sgRNA2 (unmapped reads 1) or sgRNA3 (unmapped reads 2) and those that aligned and had the correct linkage between sgRNA2 and sgRNA3 (mapped and linked). c - e , Overrepresentation analysis of the top 200 genes enriched in GFP high cell populations from the T.spiezzo ( c ), Brunello ( d ), and Cellecta ( e ) screens. Gene counts and adjusted p-value are represented in each figure. The 10 most significant GO biological processes are shown. f-m , Autophagy-related gene sets including autophagosome assembly ( GO:0000045 , n=174, f ), autophagosome membrane ( GO:0000421 , n=129, g ), autophagy of mitochondrion ( GO:0000422 , n=109, h ), autophagosome ( GO:0005776 , n=198, i ), regulation of autophagy ( GO:0010506 , n=209, j ), positive regulation of autophagy ( GO:0010508 , n=196, k ), macroautophagy ( GO:0016236 , n=180, l ), and lysosomal microautophagy ( GO:0016237 , n=6, m ) using absolute log 2 fold changes in GFP high cell populations from the T.spiezzo, Brunello, and Cellecta screens. The p value was determined by two-way ANOVA. The box plot represents the interquartile range. n , An example of flow cytometry histograms of GFP-SQSTM1 intensity in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against each of the 16 genes selected for validation or a non-targeting control (NT) lentivirus. N = 3 biological repeats. o , An example of GFP-SQSTM1 puncta in H4-Cas9-GFP-SQSTM1 cells transduced with T.spiezzo qgRNA lentivirus against each of the 16 genes selected for validation or NT controls. N = 3 biological repeats. Cells were demarcated by dashed lines according to the cytosolic GFP signal. p , Quantification of LC3II levels of cells and conditions described in Fig. . All values were normalized to the mean of the two NT repeats (- ChQ) on the same blot. Both the LC3II and normalized LC3II (LC3II/GAPDH) levels were shown to determine whether consistent changes were observed for the two biological repeats of a defined gene to determine promising candidates for further validation.

    Article Snippet: The accumulation of GFP-tagged SQSTM1 provides a reliable proxy of autophagic activity, motivating us to compare the sensitivity and specificity of a pooled T.spiezzo version with the two pooled CRISPRko libraries, Brunello and Cellecta.

    Techniques: Fluorescence, Sequencing, Membrane, Flow Cytometry, Transduction, Biomarker Discovery, Control