Journal: Cell chemical biology

Article Title: Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion.

doi: 10.1016/j.chembiol.2020.08.013

Figure Lengend Snippet: Figure 1. Recognition of the 3,849 + 10 kb C>T-induced CFTR Pseudo Exon Requires an SRSF-Dependent ESE (A–C) (A) Diagram of the CFTR splicing reporter. The CFTR minigene of exon 22–23 with a wild-type (WT reporter) or the 3,849 + 10 kb C>T-harboring (C>T reporter) IVS22 with a partial truncation (see STAR Methods for details) is fused with the amino-terminal GST and carboxyl-terminal RFP and GFP. Filled arrow, primer for RT-PCR. (B and C) Representative microscopic images of HEK293 cells transfected with the WT or C>F reporter for 24 h (B), and quantification of GFP intensity (%) from the total GFP and RFP signal (C). (D) RT-PCR for HEK293 cells transfected with the WT or C>T reporter vector for 24 h. CFTR splicing was detected by primer set oSS25 and oSS27, with the amplicon of 311 bp for the pseudo-exon-inclusion form (22/J/23) and 228 bp for the skipping form (22/23). ACTB was detected by oAM13 and oAM14, serving as a loading control. (E–G) Western blotting of phosphorylated SR proteins pulled down with biotin-conjugated RNA oligonucleotides (#1, #2, or #3 (E), shown in the diagram on the top, or #2 or #2M (G), their sequences shown in (F)) in HEK293 cell lysate. , pull-down product without bait RNA. Mutated ribonucleotides are shown in red. (H) Diagram showing the C>T and C>T(#2M) reporters. The C>T(#2M) reporter harbors point mutations of #2M shown in (F). (I) RT-PCR for HEK293 cells transfected with the WT, C>T, or C>T(#2M) reporter vector for 24 h. CFTR splicing was detected with the primer set oSS25 and oSS27. ACTB was detected by oAM13 and oAM14, serving as a loading control. (J and K) Representative microscopic images (J), and quantification analysis of percentage GFP intensity (K), for HEK293 cells transfected with the C>T or C>T(#2M) reporter for 24 h. Scale bars, 50 mm in (B and J). 22/J/23 and 22/23 stand for the pseudo exon inclusion and skipping products, respectively, in (B, D, and J). Each open circle represents the mean GFP intensity (%) of five random fields, and the mean ± SD from three independent experiments is shown in (C and K). **p < 0.01 (C and K).

Article Snippet: HEK293 cells (1.0 3 105 cells in 12-well plates) were transfected with YFP halide sensor vector by Peter Haggie (Addgene #25872, pcDNA3.1 EYFP H148Q/I152L (Galietta et al., 2001)) and CFTRWT-GFP or CFTRC>T-GFP reporter plasmid using FuGENE HD Transfection Reagent (Promega).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Amplification, Control, Western Blot

Journal: Cell chemical biology

Article Title: Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion.

doi: 10.1016/j.chembiol.2020.08.013

Figure Lengend Snippet: Figure 2. CLK Inhibitor Suppresses Inclusion of the CFTR Pseudo Exon in an ESE-Dependent Manner (A) Percentages of GFP intensity are indicated for HEK293 cells transfected with the WT or C>T reporter for 6 h and treated with the indicated compounds or DMSO (0.1%) for 19 h. (B and C) RT-PCR for HEK293 cells transfected with the WT or C>T reporter vector for 5 h and treated with the indicated compounds (B, TG003; C, SRPIN340) or DMSO (0.1%) for 19 h. CFTR splicing was detected by primers oSS25 and oSS27, and ACTB by oAM13 and oAM14. (D) Diagrams indicating the C>T, C>T-SA, and C>T(#2M)-SA reporters. In the C>T-SA reporter, the pseudo-exonic splice acceptor (SA) is replaced by the conserved SA of exon 23. The C>T(#2M)-SA vector harbors the #2M point mutation in the background of the C>T-SA vector. All three vectors harbor the 3,849 + 10 kb C>T mutation. (E and F) Representative microscopic images (E) and quantification of percentages of GFP intensity (F) for HEK293 cells transfected with the WT, C>T, C>T-SA, or C>T(#2M)-SA vector for 5 h and treated with TG003 (30 mM) or DMSO (0.1%) for 19 h. Scale bars, 50 mm in (E). Each open circle represents the mean GFP intensity (%) of five random fields, and the mean ± SD from three independent experiments is shown in (A and F). **p < 0.01. 22/J/23 and 22/23 stand for the pseudo exon inclusion and skipping products, respectively in (B, C, and E).

Article Snippet: HEK293 cells (1.0 3 105 cells in 12-well plates) were transfected with YFP halide sensor vector by Peter Haggie (Addgene #25872, pcDNA3.1 EYFP H148Q/I152L (Galietta et al., 2001)) and CFTRWT-GFP or CFTRC>T-GFP reporter plasmid using FuGENE HD Transfection Reagent (Promega).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Mutagenesis

Journal: Cell chemical biology

Article Title: Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion.

doi: 10.1016/j.chembiol.2020.08.013

Figure Lengend Snippet: Figure 3. Identification of CaNDY as a Highly Potent Agent in Suppressing the 3,849 + 10 kb C>T-Induced Pseudo Exon of CFTR (A) Scatterplot for the recovery rates of CFTR exon 22/23 splicing in the focused library screening of CLK inhibitor analogs. (B) Structure of CaNDY. (C) Sigmoidal dose-response curve for pseudo-exon-suppression activity of CaNDY and TG003 (0.1, 0.3, 1, 3, 10, or 30 mM) in HEK293 cells transfected with the WT or C>T reporter and treated with the compounds for 13 h. (D and E) RT-PCR analysis for HEK293 (D) and Calu-3 cells (E) transfected with the WT or C>T reporter for 5 h and treated with CaNDY (1, 3, or 10 mM) or DMSO (0.1%) for 19 h. Untransfected cells were also analyzed as a negative control (No TF). Exogenous CFTR was detected by oSS25 and oSS27, and endogenous CFTR was detected by oAM619 and oSS30 (yielding a 430-bp product for pseudo exon inclusion and a 346-bp product for pseudo exon skipping). Rates of normal CFTR splicing (% 22/23) were calculated from the band intensities. ND indicates there was no detectable product after 35 cycles of PCR amplification. (F) RT-PCR analysis for B lymphocyte GM11860 with CaNDY (1, 3, or 10 mM), DMSO (0.1%), or cycloheximide (CHX) (100 mg/mL) for 8 h. Endogenous CFTR was detected with the primers oSS29 and oSS30, yielding a 340-bp product for pseudo exon inclusion and a 256-bp product for pseudo exon skipping. Identities of the CFTR splicing products were confirmed by Sanger sequencing.

Article Snippet: HEK293 cells (1.0 3 105 cells in 12-well plates) were transfected with YFP halide sensor vector by Peter Haggie (Addgene #25872, pcDNA3.1 EYFP H148Q/I152L (Galietta et al., 2001)) and CFTRWT-GFP or CFTRC>T-GFP reporter plasmid using FuGENE HD Transfection Reagent (Promega).

Techniques: Library Screening, Activity Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Negative Control, Sequencing

Journal: Cell chemical biology

Article Title: Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion.

doi: 10.1016/j.chembiol.2020.08.013

Figure Lengend Snippet: Figure 4. CaNDY, but Not CFTR Modulator VX-809, Rescues Mature CFTR Expression from the CFTR Gene with the 3,849 + 10 kb C>T mu- tation (A) Diagram of HiBiTWT and HiBiTC>T expression vectors. Upon translation, cleavage at the 2A site releases amino-terminal EGFP from the full-length or truncated CFTR with or without HiBiT-HA, respectively. (B and C) HEK293 cells were transfected with HiBiTWT or HiBiTC>T for 5 h and treated with CaNDY (1 or 3 mM) or DMSO (0.1%) for 48 h. Full-length expression of CFTR was quantified by relative luminescence units (RLU) generated by the C-terminal HiBiT tag using the Nano-Glo HiBiT Lytic Detection System (B), and expression of EGFP was confirmed by western blotting (C). Columns indicate mean ± SD for independent experiments (n = 3, indicated individually by circles). (D) Structures of CFTRWT-GFP, CFTRC>T-GFP, and CFTRF508d-GFP expression vectors are shown. CFTRWT-GFP and CFTRC>T-GFP vectors harbor a partial IVS22 of wild type or with the 3,849 + 10 kb C>T mutation, respectively, and CFTRF508d-GFP expresses the F508 deletion form of CFTR. In these constructs, mKate2 with the 2A site is fused amino-terminally and AcGFP carboxyl-terminally with the CFTR coding sequence, and inclusion of the pseudo exon results in CFTR truncation without AcGFP translation.

Article Snippet: HEK293 cells (1.0 3 105 cells in 12-well plates) were transfected with YFP halide sensor vector by Peter Haggie (Addgene #25872, pcDNA3.1 EYFP H148Q/I152L (Galietta et al., 2001)) and CFTRWT-GFP or CFTRC>T-GFP reporter plasmid using FuGENE HD Transfection Reagent (Promega).

Techniques: Expressing, Transfection, Generated, Western Blot, Mutagenesis, Construct, Sequencing

Journal: Molecular cell

Article Title: Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

doi: 10.1016/j.molcel.2019.09.012

Figure Lengend Snippet: Figure 1. The AcrIIA6 Binding Mode to St1Cas9 Surveillance Complex Revealed by Cryo-EM (A) Interactions between AcrIIA6 and apo-St1Cas9 (St1Cas9) or St1Cas9 RNP (St1Cas9,sgRNA) were monitored using BLi. AcrIIA6 stably binds to the St1Cas9 RNP, as shown by their slow dissociation rate, but does not bind to apo-St1Cas9. The binding curves of negative controls apo-SpCas9 (SpCas9) and SpCas9 RNP (SpCas9,sgRNA) are shown in the inset. Experiments were performed three times and yielded equivalent results. One experiment is shown. (B) Representative cryo-EM 2D class averages of monomeric (box size: 263 A˚ 3 263 A˚ ) and dimeric (box size: 334 A˚ 3 334 A˚ ) assemblies of tDNA20-bound St1Cas9 RNP in complex with AcrIIA6. (C and D) Orthogonal views of cryo-EM 3D reconstructions of the monomeric (C) and dimeric (D) assemblies. See also Figures S1, S2, and S3.

Article Snippet: The U6-driven sgRNA expression plasmid for St1Cas9 (St1Cas9_LMD-9_sgRNA_pUC19; Addgene #110627) was synthesized as a gBlock gene fragment (Integrated DNA Technologies) and cloned into pUC19.

Techniques: Binding Assay, Cryo-EM Sample Prep, Stable Transfection

Journal: Molecular cell

Article Title: Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

doi: 10.1016/j.molcel.2019.09.012

Figure Lengend Snippet: Figure 2. Structural Overview of St1Cas9 and Its sgRNA (A) Domain organization of St1Cas9. BH: bridge helix, CTD: C-terminal domain, PI: PAM-interacting domain, WED: wedge domain. (B) Sequences of the sgRNA (SL1: stem loop 1, SL2: stem loop 2) and tDNA20. Grey nucleotides in the target-guide heteroduplex, repeat-antirepeat duplex, and SL2 were not be modeled. The G1 nucleotide in the in vitro transcribed sgRNA substitutes the theoretical C1 nucleotide. Watson-Crick and G:U wobble base pairs are shown as lines and filled circles, respectively. Green boxes indicate conserved structural features in CRISPR-Cas9 systems. (C) Surface and ribbon representations of the St1Cas9,sgRNA,tDNA20 complex (AcrIIA6 is not shown for clarity). The linker between the REC and NUC lobes (457–463) is not modeled. The back face of St1Cas RNP, relative to the DNA binding crevasse, is shown on the left-hand side. See also Figure S4.

Article Snippet: The U6-driven sgRNA expression plasmid for St1Cas9 (St1Cas9_LMD-9_sgRNA_pUC19; Addgene #110627) was synthesized as a gBlock gene fragment (Integrated DNA Technologies) and cloned into pUC19.

Techniques: In Vitro, CRISPR, Binding Assay

Journal: Molecular cell

Article Title: Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

doi: 10.1016/j.molcel.2019.09.012

Figure Lengend Snippet: Figure 3. AcrIIA6 Binds to an Allosteric Site on St1Cas9 (A) Surface representation of the monomeric inhibition complex showing the AcrIIA6 dimer bound to an allosteric site at the back face of St1Cas9, relative to the target-binding crevasse and catalytic domains. Orthogonal views of the front (left), side (middle) and back (right) of the complex. The intermolecular interactions between St1Cas9,sgRNA and the b2-b3 hairpin (d), L8 loop (-) and L9 loop (A) of AcrIIA6 subunit A, and a5-a6 helices (+) of AcrIIA6 subunit B are indicated. (B) Surface representation of the symmetric dimeric inhibition complex. AcrIIA6 dimer contains two identical St1Cas9-binding sites, each one built by regions of both AcrIIA6 subunits A and B. (C–F) Close-up views of the intermolecular interactions between St1Cas9,sgRNA and AcrIIA6 subunit A b2-b3 hairpin (d) (C), L8 loop (-) (D), L9 loop (A) (E), and subunit B a5-a6 helices (+) (F). Hydrogen bonds are shown as black dotted lines, and the salt bridge is shown as a red dotted line. See also Figure S4.

Article Snippet: The U6-driven sgRNA expression plasmid for St1Cas9 (St1Cas9_LMD-9_sgRNA_pUC19; Addgene #110627) was synthesized as a gBlock gene fragment (Integrated DNA Technologies) and cloned into pUC19.

Techniques: Inhibition, Binding Assay

Journal: Molecular cell

Article Title: Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

doi: 10.1016/j.molcel.2019.09.012

Figure Lengend Snippet: Figure 4. AcrIIA6 Reduces St1Cas9 Surveillance Complex DNA Binding Affinity and Blocks DNA Binding in Cells (A) Time-course cleavage assay using dsDNA fragments containing a target sequence and a PAM motif. St1Cas9 and AcrIIAs were mixed with a 1:1 molar ratio (13). Mobilities of input DNA (uncleaved) and cleavage products (cleaved) are indicated with arrows. AcrIIA6 inhibits St1Cas9,sgRNA-mediated DNA cleavage. The negative control AcrIIA2 does not affect St1Cas9 activity. Experiments were performed three times and yielded equivalent results. One experiment is shown. (B) Interactions between dsDNA fragments (59-bp molecules containing the target and PAM sequences) and pre-formed St1Cas9 RNP or AcrIIAs-bound St1Cas9 RNP were monitored using BLi. St1Cas9 and AcrIIAs were mixed with 1:1 (13) or 1:2 (23) molar ratios. AcrIIA6 markedly reduces the St1Cas9 RNP DNA binding affinity. Experiments were performed three times and yielded equivalent results. One experiment is shown. (C) Quantification of base editing mediated by St1BE4max LMD-9 when co-expressed in presence of the indicated AcrIIAs in K562 cells. Empty pVAX backbone was used as a negative control. Guide sequences are shown with target cytosines highlighted in blue. Dashes indicate that base editing was not detected. Experiments were performed twice for each guide and AcrIIA combinations and yielded equivalent results. One experiment is shown. See also Figure S5.

Article Snippet: The U6-driven sgRNA expression plasmid for St1Cas9 (St1Cas9_LMD-9_sgRNA_pUC19; Addgene #110627) was synthesized as a gBlock gene fragment (Integrated DNA Technologies) and cloned into pUC19.

Techniques: Binding Assay, Cleavage Assay, Sequencing, Negative Control, Activity Assay

Journal: Molecular cell

Article Title: Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

doi: 10.1016/j.molcel.2019.09.012

Figure Lengend Snippet: Figure 5. AcrIIA6 Alters St1Cas9 Conformational Dynamics Associated with PAM Binding (A) Representative cryo-EM 2D class averages showing the absence or presence of AcrIIA6 (marked by purple arrowhead) (box size: 263 A˚ 3 263 A˚ ). (B and C) Orthogonal views of cryo-EM 3D reconstructions of St1Cas9,sgRNA,tDNA59-ntPAM complex (B) and its AcrIIA6-bound form (C). (D) (Left) Ribbon representation of the 3D structures of St1Cas9,sgRNA,AcrIIA6,tDNA59-ntPAM (color-coded) superimposed onto the 3D structure of St1Cas9,sgRNA,tDNA20,AcrIIA6 (light blue). (Right) Close-up view of the PAM binding site. The arrows indicate the PAM binding-induced shifts in the St1Cas9 WED and PI domains and in the AcrIIA6 dimer. (E) (Left) Ribbon representation of the 3D structures of St1Cas9,sgRNA,AcrIIA6,tDNA59-ntPAM (color-coded) superimposed onto the 3D structure of of St1Cas9,sgRNA,tDNA59-ntPAM (khaki). (Right) Close-up view of the PAM binding site. The arrows indicate the movement of the St1Cas WED domain in the AcrIIA6-free St1Cas9,sgRNA,tDNA59-ntPAM complex. See also Figures S6 and S7.

Article Snippet: The U6-driven sgRNA expression plasmid for St1Cas9 (St1Cas9_LMD-9_sgRNA_pUC19; Addgene #110627) was synthesized as a gBlock gene fragment (Integrated DNA Technologies) and cloned into pUC19.

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Molecular cell

Article Title: Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

doi: 10.1016/j.molcel.2019.09.012

Figure Lengend Snippet: Figure 6. A Natural Variant of St1Cas9 Is Resistant to AcrIIA6 Inhibition (A) Alignment of the C terminus of St1Cas9 proteins isolated from S. thermophilus strains LMD-9, CNRZ 1066, and LMG 18311. (B) Indel frequencies mediated by the different St1Cas9s when co-expressed in presence of the indicated AcrIIAs in K562 cells. Indels were analyzed by TIDE assay. Empty pVAX backbone was used as a negative control. Experiments were performed twice for each guide, AcrIIA, and St1Cas9 combinations and yielded equivalent results. One experiment is shown. (C) Surface and ribbon representations of the intermolecular contacts between St1Cas9 and AcrIIA6 that are impaired by the amino acid substitutions identified in the TOPO domain of St1Cas9 LMG 18311 (K1001E, K1008M, and G993K). E1010 and M1008 would disrupt hydrogen bonds (dotted lines) and introduce repulsive forces, while K993 would cause steric hindrance.

Article Snippet: The U6-driven sgRNA expression plasmid for St1Cas9 (St1Cas9_LMD-9_sgRNA_pUC19; Addgene #110627) was synthesized as a gBlock gene fragment (Integrated DNA Technologies) and cloned into pUC19.

Techniques: Variant Assay, Inhibition, Isolation, Negative Control, Introduce

Journal: ACS Synthetic Biology

Article Title: A Fluorescent Split Aptamer for Visualizing RNA–RNA Assembly In Vivo

doi: 10.1021/acssynbio.7b00059

Figure Lengend Snippet: The Split-Broccoli system functions when expressed in vivo . (a) DNA templates corresponding to 3WJdB-T (black), Top-T (yellow), Bottom-T (blue) were individually cloned into the pUC19 plasmid. A single plasmid expressing both Top-T and Bottom-T was created (pUC19-P70a-Top-T∼P70a-Bottom-T), as was a control plasmid for runon transcription which lacked a promoter immediately upstream of Bottom-T (pUC19-P70a-Top-T∼Bottom-T). (b) A representative flow cytometry histogram of 5 × 10 4 events per population illustrates a shift in fluorescence for the plasmid containing the Split-Broccoli expression plasmid (green). Bacterial populations transformed with plasmids containing either Top-T or Bottom-T alone, or lacking a promoter upstream of Bottom-T , demonstrate background levels of fluorescence equivalent to the unmodified pUC19 plasmid control. (c) Relative mean fluorescence intensities for flow cytometric analyses of transformed populations, normalized to the pUC19 plasmid (set to 0) and 3WJdB-T (set to 1), are shown with error bars to indicate standard deviations ( n ≥ 4). (d) Fluorescence microscopy imaging further validates the in vivo functionality of the Split-Broccoli system, as green fluorescence is only observed for E. coli transformed with either the unimolecular 3WJdB-T encoding plasmid or bimolecular Split-Broccoli encoding plasmid.

Article Snippet: Plasmids for in vivo transcription by native E. coli RNA polymerase were deposited to Addgene (IDs 87311–87315).

Techniques: In Vivo, Clone Assay, Plasmid Preparation, Expressing, Control, Flow Cytometry, Fluorescence, Transformation Assay, Microscopy, Imaging

Journal: ACS Synthetic Biology

Article Title: A Fluorescent Split Aptamer for Visualizing RNA–RNA Assembly In Vivo

doi: 10.1021/acssynbio.7b00059

Figure Lengend Snippet: Split-Broccoli is modular and can be used to monitor RNA–RNA hybridization events in vivo . (a) A Split-Broccoli Toehold Switch plasmid was constructed to include two constitutively expressed transcription units. The first transcription unit encodes Top (yellow) and Toehold (gray) sequences within the 5′ UTR of the mCherry mRNA (red). Translation of the Top-Toehold-mCherry mRNA is suppressed due to sequestration of the ribosome binding site (orange) and start codon within the toehold structure (boxed). The second transcription unit encodes Trigger (gray) and Bottom (blue) sequences, which can base pair with Top-Toehold-mCherry . (b) Hybridization of Top-Toehold-mCherry with Trigger-Bottom allows fluorescence activation of the Split-Broccoli system and translation of mCherry . (c) Green fluorescence (left columns) and red fluorescence (right columns) from flow cytometric analysis of populations show background levels of fluorescence for plasmids encoding a single transcription unit only ( Top-Toehold-mCherry or Trigger-Bottom ). Top + Bottom , which transcribes the Split-Broccoli system, exhibits only green fluorescence, while the Split-Broccoli Toehold Switch plasmid ( Top-Toehold-mCherry + Trigger-Bottom ) exhibits both red and green fluorescence, indicating both hybridization of Split-Broccoli and translation of mCherry . Grand mean fluorescence intensity ( n = 4) is shown with error bars to indicate standard deviations. (d) Fluorescence microscopy imaging of E. coli harboring the Split-Broccoli Toehold Switch plasmid confirms hybridization of the Top and Bottom components of Split-Broccoli (green fluorescence) and activation of mCherry translation (red fluorescence). (e) An E. coli cell-free system (TX-TL) was used to monitor transcription and translation of the Split-Broccoli Toehold Switch plasmid and demonstrates the increased temporal sensitivity of Split-Broccoli (green fluorescence, left axis) over mCherry (red fluorescence, right axis). Mean values ( n = 3) are shown with error bars to indicate standard deviations.

Article Snippet: Plasmids for in vivo transcription by native E. coli RNA polymerase were deposited to Addgene (IDs 87311–87315).

Techniques: Hybridization, In Vivo, Plasmid Preparation, Construct, Binding Assay, Fluorescence, Activation Assay, Microscopy, Imaging