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( A ) Inducible gRNA barcode design: A spacer-blocking hairpin structure is engineered to include a 15-base semirandom genetic barcode within the hairpin loop. In addition, an 8A8G poly(A) tail is appended at the 3′ end of the barcoding gRNA. ( B ) Oligo-CALL system constructs: (i) Dual gRNA barcoding construct: two distinct barcoding gRNA, each driven by human or mouse U6 promoters, separately, with Thy1.1 as a constitutive selection marker. (ii) The green fluorescent protein (GFP) reporter construct: contains three tandem repeats of the barcoding gRNA target site upstream of a miniCMV (minimal cytomegalovirus) promoter controlling GFP expression; blue fluorescent protein (BFP) serves as a selection marker. (iii) CRISPRa construct: constitutively expresses dCas9-VPR [deactivated Cas9 fused to a transcriptional activator VPR (VP64, p65, and Rta)] and the red fluorescent protein (RFP; Cherry variant). All constructs use the <t>lentiviral</t> backbone to facilitate efficient cell transduction. hU6, human U6; mU6, mouse U6; EF1α, elongation factor-1 alpha promoter. ( C ) GFP activation with Oligo-CALL system: In the default state, a self-folding hairpin within the gRNA spacer prevents CRISPRa from activating GFP expression. Introduction of BC-ASOs disrupts this hairpin, exposing the gRNA spacer and enabling robust GFP expression. Representative fluorescence images show strong GFP induction following BC-ASO transfection, in contrast to minimal activation in cells receiving no ASO or mismatched ASOs (mis ASOs). ( D to F ) Flow cytometry analyses of GFP activation in Oligo-CALL–labeled cells: H358 cells bearing BC1 barcodes were transfected with ASOs against BC1. Flow cytometry shows the percentage of GFP + cells (D and E) and the mean fluorescent intensity (F) across the overall population (48 hours post-ASO transfection; n = 2 biological replicates). MFI, mean fluorescence intensity. Cont., control.
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( A ) Inducible gRNA barcode design: A spacer-blocking hairpin structure is engineered to include a 15-base semirandom genetic barcode within the hairpin loop. In addition, an 8A8G poly(A) tail is appended at the 3′ end of the barcoding gRNA. ( B ) Oligo-CALL system constructs: (i) Dual gRNA barcoding construct: two distinct barcoding gRNA, each driven by human or mouse U6 promoters, separately, with Thy1.1 as a constitutive selection marker. (ii) The green fluorescent protein (GFP) reporter construct: contains three tandem repeats of the barcoding gRNA target site upstream of a miniCMV (minimal cytomegalovirus) promoter controlling GFP expression; blue fluorescent protein (BFP) serves as a selection marker. (iii) CRISPRa construct: constitutively expresses dCas9-VPR [deactivated Cas9 fused to a transcriptional activator VPR (VP64, p65, and Rta)] and the red fluorescent protein (RFP; Cherry variant). All constructs use the <t>lentiviral</t> backbone to facilitate efficient cell transduction. hU6, human U6; mU6, mouse U6; EF1α, elongation factor-1 alpha promoter. ( C ) GFP activation with Oligo-CALL system: In the default state, a self-folding hairpin within the gRNA spacer prevents CRISPRa from activating GFP expression. Introduction of BC-ASOs disrupts this hairpin, exposing the gRNA spacer and enabling robust GFP expression. Representative fluorescence images show strong GFP induction following BC-ASO transfection, in contrast to minimal activation in cells receiving no ASO or mismatched ASOs (mis ASOs). ( D to F ) Flow cytometry analyses of GFP activation in Oligo-CALL–labeled cells: H358 cells bearing BC1 barcodes were transfected with ASOs against BC1. Flow cytometry shows the percentage of GFP + cells (D and E) and the mean fluorescent intensity (F) across the overall population (48 hours post-ASO transfection; n = 2 biological replicates). MFI, mean fluorescence intensity. Cont., control.
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( A ) Inducible gRNA barcode design: A spacer-blocking hairpin structure is engineered to include a 15-base semirandom genetic barcode within the hairpin loop. In addition, an 8A8G poly(A) tail is appended at the 3′ end of the barcoding gRNA. ( B ) Oligo-CALL system constructs: (i) Dual gRNA barcoding construct: two distinct barcoding gRNA, each driven by human or mouse U6 promoters, separately, with Thy1.1 as a constitutive selection marker. (ii) The green fluorescent protein (GFP) reporter construct: contains three tandem repeats of the barcoding gRNA target site upstream of a miniCMV (minimal cytomegalovirus) promoter controlling GFP expression; blue fluorescent protein (BFP) serves as a selection marker. (iii) CRISPRa construct: constitutively expresses dCas9-VPR [deactivated Cas9 fused to a transcriptional activator VPR (VP64, p65, and Rta)] and the red fluorescent protein (RFP; Cherry variant). All constructs use the <t>lentiviral</t> backbone to facilitate efficient cell transduction. hU6, human U6; mU6, mouse U6; EF1α, elongation factor-1 alpha promoter. ( C ) GFP activation with Oligo-CALL system: In the default state, a self-folding hairpin within the gRNA spacer prevents CRISPRa from activating GFP expression. Introduction of BC-ASOs disrupts this hairpin, exposing the gRNA spacer and enabling robust GFP expression. Representative fluorescence images show strong GFP induction following BC-ASO transfection, in contrast to minimal activation in cells receiving no ASO or mismatched ASOs (mis ASOs). ( D to F ) Flow cytometry analyses of GFP activation in Oligo-CALL–labeled cells: H358 cells bearing BC1 barcodes were transfected with ASOs against BC1. Flow cytometry shows the percentage of GFP + cells (D and E) and the mean fluorescent intensity (F) across the overall population (48 hours post-ASO transfection; n = 2 biological replicates). MFI, mean fluorescence intensity. Cont., control.
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( A ) Inducible gRNA barcode design: A spacer-blocking hairpin structure is engineered to include a 15-base semirandom genetic barcode within the hairpin loop. In addition, an 8A8G poly(A) tail is appended at the 3′ end of the barcoding gRNA. ( B ) Oligo-CALL system constructs: (i) Dual gRNA barcoding construct: two distinct barcoding gRNA, each driven by human or mouse U6 promoters, separately, with Thy1.1 as a constitutive selection marker. (ii) The green fluorescent protein (GFP) reporter construct: contains three tandem repeats of the barcoding gRNA target site upstream of a miniCMV (minimal cytomegalovirus) promoter controlling GFP expression; blue fluorescent protein (BFP) serves as a selection marker. (iii) CRISPRa construct: constitutively expresses dCas9-VPR [deactivated Cas9 fused to a transcriptional activator VPR (VP64, p65, and Rta)] and the red fluorescent protein (RFP; Cherry variant). All constructs use the lentiviral backbone to facilitate efficient cell transduction. hU6, human U6; mU6, mouse U6; EF1α, elongation factor-1 alpha promoter. ( C ) GFP activation with Oligo-CALL system: In the default state, a self-folding hairpin within the gRNA spacer prevents CRISPRa from activating GFP expression. Introduction of BC-ASOs disrupts this hairpin, exposing the gRNA spacer and enabling robust GFP expression. Representative fluorescence images show strong GFP induction following BC-ASO transfection, in contrast to minimal activation in cells receiving no ASO or mismatched ASOs (mis ASOs). ( D to F ) Flow cytometry analyses of GFP activation in Oligo-CALL–labeled cells: H358 cells bearing BC1 barcodes were transfected with ASOs against BC1. Flow cytometry shows the percentage of GFP + cells (D and E) and the mean fluorescent intensity (F) across the overall population (48 hours post-ASO transfection; n = 2 biological replicates). MFI, mean fluorescence intensity. Cont., control.

Journal: Science Advances

Article Title: Oligo-CALL: A next-generation barcoding platform for studying resistance to targeted therapy

doi: 10.1126/sciadv.adw9990

Figure Lengend Snippet: ( A ) Inducible gRNA barcode design: A spacer-blocking hairpin structure is engineered to include a 15-base semirandom genetic barcode within the hairpin loop. In addition, an 8A8G poly(A) tail is appended at the 3′ end of the barcoding gRNA. ( B ) Oligo-CALL system constructs: (i) Dual gRNA barcoding construct: two distinct barcoding gRNA, each driven by human or mouse U6 promoters, separately, with Thy1.1 as a constitutive selection marker. (ii) The green fluorescent protein (GFP) reporter construct: contains three tandem repeats of the barcoding gRNA target site upstream of a miniCMV (minimal cytomegalovirus) promoter controlling GFP expression; blue fluorescent protein (BFP) serves as a selection marker. (iii) CRISPRa construct: constitutively expresses dCas9-VPR [deactivated Cas9 fused to a transcriptional activator VPR (VP64, p65, and Rta)] and the red fluorescent protein (RFP; Cherry variant). All constructs use the lentiviral backbone to facilitate efficient cell transduction. hU6, human U6; mU6, mouse U6; EF1α, elongation factor-1 alpha promoter. ( C ) GFP activation with Oligo-CALL system: In the default state, a self-folding hairpin within the gRNA spacer prevents CRISPRa from activating GFP expression. Introduction of BC-ASOs disrupts this hairpin, exposing the gRNA spacer and enabling robust GFP expression. Representative fluorescence images show strong GFP induction following BC-ASO transfection, in contrast to minimal activation in cells receiving no ASO or mismatched ASOs (mis ASOs). ( D to F ) Flow cytometry analyses of GFP activation in Oligo-CALL–labeled cells: H358 cells bearing BC1 barcodes were transfected with ASOs against BC1. Flow cytometry shows the percentage of GFP + cells (D and E) and the mean fluorescent intensity (F) across the overall population (48 hours post-ASO transfection; n = 2 biological replicates). MFI, mean fluorescence intensity. Cont., control.

Article Snippet: Lentiviral backbone vectors—Dual-sgRNA (plasmid #154194), CaTCH_empty (plasmid #157746), and dCas9-VPR_P2A_mCherry (plasmid #154193), developed initially in A. Obenauf’s laboratory ( )—were obtained from Addgene.

Techniques: Blocking Assay, Construct, Selection, Marker, Expressing, Variant Assay, Transduction, Activation Assay, Fluorescence, Transfection, Flow Cytometry, Labeling, Control