Journal: Nature Communications

Article Title: PAM-flexible genome editing with an engineered chimeric Cas9

doi: 10.1038/s41467-023-41829-y

Figure Lengend Snippet: A Quantitative analysis of indel formation with indicated Cas9 variants. Indel frequencies were determined via batch analysis following PCR amplification of indicated genomic loci, in comparison to unedited controls for each gene target. All samples were performed in independent transfection replicates and the mean of the quantified indel formation values was calculated. All gRNA sequences can be found in Supplementary Table . B Quantitative analysis of A-to-G with indicated ABE8e variants. Base editing conversion rates were determined via BEEP following PCR amplification of indicated genomic loci, in comparison to unedited controls for each gene target. All samples were performed in independent transfection replicates and the mean of the quantified base editing formation values was calculated. All gRNA sequences can be found in Supplementary Table . C Off-targets as identified by GUIDE-seq genome-wide for SpCas9, Sc + +, SpRY, and SpRYc each paired with two sgRNAs targeting either EMX1 or VEGFA . Only sites that harbored a sequence with ≤10 mismatches relative to the gRNA were considered potential off-target sites. D Efficiency heatmap of mismatch tolerance assay on genomic targets. Quantified indel frequencies are exhibited for each labeled single or double mismatch (number of bases 5’ upstream of the PAM) in the sgRNA sequence for the indicated Cas9 variant and indicated PAM sequence. All samples were performed in independent transfection replicates and the mean of the quantified indel formation values was calculated.

Article Snippet: 180 ng of PAM library (Addgene #160132) was incubated with 30 nM of sgRNA and 6 μL of fluorescein-normalized lysate.

Techniques: Amplification, Comparison, Transfection, Genome Wide, Sequencing, Labeling, Variant Assay

Journal: Nature Communications

Article Title: PAM-flexible genome editing with an engineered chimeric Cas9

doi: 10.1038/s41467-023-41829-y

Figure Lengend Snippet: A Schematic of SpRYc MECP2 cell line generation and SpRYc-ABE8e editing. Figure was created with BioRender.com. B Base editing conversion rates were determined via CRISPResso2 NGS analysis following PCR amplification of MECP2 -integrated loci, in comparison to SpCas9-ABE8e and SpRYc-ABE8e for the C502T installed mutation. Samples were performed in independent nucleofection triplicates ( n = 3) ± SD, with the center of error bars depicting the mean. For individual samples, statistical significance was determined by a two-tailed Student’s t test, as compared to the SpCas9-ABE8e control. The exact p values for SpRY-ABE8e and SpRYc-ABE8e compared to SpCas9-ABE8e are both <0.0001. Calculated p values are represented as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. C SpRYc-BE4Max was nucleofected into TruHD cells alongside an sgRNA targeting the HTT repeat. Base editing conversion rate was determined via CRISPResso2 NGS analysis following PCR amplification of the HTT loci. Samples were performed in independent nucleofection triplicates ( n = 3) ± SD, with the center of error bars depicting the mean. For individual samples, statistical significance was determined by a two-tailed Student’s t test, as compared to the SpCas9 control. The exact p values for SpRY-BE4Max and SpRYc-BE4Max compared to S p Cas9-BE4Max are 0.001 and 0.0028, respectively. Calculated p values are represented as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. The sgRNA and PAM sequences are annotated within their relative positions to the CAG repeat. The red annotation indicates the base to be mutated. The figure was made via Geneious Prime 2023.1.2. D Structural insights via homology modeling in SWISS-MODEL. (i) Interaction of the engineered Sc + + loop (purple) with the backbone of the target strand (TS) PAM region. The REC1 loop from wild-type SpCas9 is indicated in green. (ii) Potential interaction of residue R1331 with the non-target strand (NTS) backbone. (iii) Multiple mutations within the PAM interaction loop allow for a more flexible PAM readout. (iv) The potential van der Waals interaction of W1145 with the ribose moieties of nontarget strand residues could further stabilize the PAM interaction.

Article Snippet: 180 ng of PAM library (Addgene #160132) was incubated with 30 nM of sgRNA and 6 μL of fluorescein-normalized lysate.

Techniques: Amplification, Comparison, Mutagenesis, Two Tailed Test, Control, Residue

Journal: Nature Communications

Article Title: How Leiomodin and Tropomodulin use a common fold for different actin assembly functions

doi: 10.1038/ncomms9314

Figure Lengend Snippet: ( a ) Domain organization of Tmod1 and Lmods, and design of the Tmod1–Lmod2 c hybrid construct. Numbers under the diagrams indicate the boundaries of domains. For Tmod1, the helix of ABS1 (aa 67–81) and the LRR portion of ABS2 (aa 181–337) are highlighted. ( b ) Nucleation activity of full-length Lmod1, Lmod2 and the hybrid construct Tmod1–Lmod2 C as compared with Tmod1 and the Arp2/3 complex (25 nM, activated by 100 nM N-WASP WCA). The left two graphs show time courses of polymerization of 2 μM Mg–ATP–actin (6% pyrene labelled) in the presence of 25 nM of the indicated proteins (colour coded) or the buffer control (black). The graph on the right shows the concentration dependence of the polymerization rates, displayed as the mean of three experiments±s.e.m. ( c , d ) Contribution of the various domains of Lmod1 ( c ) and Lmod2 ( d ) to the nucleation activity. The graphs on the left and the right show, respectively, the time course of polymerization of 2 μM Mg–ATP–actin in the presence of 25 nM Lmod fragments (colour coded) or buffer (black) and the concentration dependence of polymerization rates.

Article Snippet: For expression in mammalian cells, Lmod and Tmod constructs, including Tmod1 N (1–163), Tmod1 ABS2 (164–351), Lmod2 N (1–179), Lmod2 ABS2 (180–367), Lmod1 N (1–298) and Lmod1 ABS2 (299–486), were cloned between the XhoI and BamHI sites of vector pEGFP-N1 (Clontech) or NheI and KpnI sites of vector mCherry2-N1 (Addgene plasmid # 54517).

Techniques: Construct, Activity Assay, Control, Concentration Assay

Journal: Nature Communications

Article Title: How Leiomodin and Tropomodulin use a common fold for different actin assembly functions

doi: 10.1038/ncomms9314

Figure Lengend Snippet: ( a ) Sequence conservation analysis of Tmod and Lmod (see also ). Fifty Tmod (purple) and fifty Lmod (orange) sequences were aligned separately or together (blue), and residue conservation scores were calculated with the program Scorecons and plotted on the human Tmod1 sequence (the scores of residues absent in Tmod1 are not shown). The Tmod1 diagram on top indicates the boundaries of the TM- and actin-binding sites. Diagrams on the bottom illustrate ABS1 constructs, and hybrid Tmod1 (magenta)/Lmod (green) ABS2 constructs (TL1 ABS2 , TL2 ABS2 and Tmod1 ABS2 Mut). The 11 residues of Tmod1 ABS2 replaced by their Lmod1 counterparts (highlighted in green across the conservation plots) tend to be conserved among Lmods, but poorly conserved between Lmods and Tmods. ( b ) Concentration dependence of polymerization rates of Lmod1 and Lmod2 in the absence (solid lines) or the presence (broken lines) of 1 μM TM, displayed as the mean of three experiments±s.e.m. ( c ) ITC titrations of ABS1 constructs (as indicated) into LatB-actin. The experimental conditions are listed for each experiment, including temperature and the concentrations of ABS1 constructs in the syringe and LatB-actin in the cell. Open symbols correspond to titrations into buffer. Only the titration of Tmod1 ABS1 could be fitted to a binding isotherm (red curve, fitting parameters inside graph), whereas Lmod1 ABS1 and Lmod2 ABS1 did not appear to bind (solid black symbols). Errors correspond to the s.d. of the fits.

Article Snippet: For expression in mammalian cells, Lmod and Tmod constructs, including Tmod1 N (1–163), Tmod1 ABS2 (164–351), Lmod2 N (1–179), Lmod2 ABS2 (180–367), Lmod1 N (1–298) and Lmod1 ABS2 (299–486), were cloned between the XhoI and BamHI sites of vector pEGFP-N1 (Clontech) or NheI and KpnI sites of vector mCherry2-N1 (Addgene plasmid # 54517).

Techniques: Sequencing, Residue, Binding Assay, Construct, Concentration Assay, Titration

Journal: Nature Communications

Article Title: How Leiomodin and Tropomodulin use a common fold for different actin assembly functions

doi: 10.1038/ncomms9314

Figure Lengend Snippet: ( a ) Time courses of polymerization and concentration dependence of polymerization rates of ABS2 constructs (as indicated) compared with full-length Lmod1 and Lmod2 (colour coded). Experimental conditions listed on top. ( b ) ITC titrations of ABS2 constructs (as indicated) into LatB-actin. The experimental conditions and fitting parameters are listed with each experiment. All the titrations fitted to a two-binding-site model (see also ). Open symbols correspond to titrations into buffer. Errors correspond to the s.d. of the fits.

Article Snippet: For expression in mammalian cells, Lmod and Tmod constructs, including Tmod1 N (1–163), Tmod1 ABS2 (164–351), Lmod2 N (1–179), Lmod2 ABS2 (180–367), Lmod1 N (1–298) and Lmod1 ABS2 (299–486), were cloned between the XhoI and BamHI sites of vector pEGFP-N1 (Clontech) or NheI and KpnI sites of vector mCherry2-N1 (Addgene plasmid # 54517).

Techniques: Concentration Assay, Construct, Binding Assay

Journal: Nature Communications

Article Title: How Leiomodin and Tropomodulin use a common fold for different actin assembly functions

doi: 10.1038/ncomms9314

Figure Lengend Snippet: Crystallographic data and refinement statistics.

Article Snippet: For expression in mammalian cells, Lmod and Tmod constructs, including Tmod1 N (1–163), Tmod1 ABS2 (164–351), Lmod2 N (1–179), Lmod2 ABS2 (180–367), Lmod1 N (1–298) and Lmod1 ABS2 (299–486), were cloned between the XhoI and BamHI sites of vector pEGFP-N1 (Clontech) or NheI and KpnI sites of vector mCherry2-N1 (Addgene plasmid # 54517).

Techniques: Solvent

Journal: Nature Communications

Article Title: How Leiomodin and Tropomodulin use a common fold for different actin assembly functions

doi: 10.1038/ncomms9314

Figure Lengend Snippet: ( a ) Superimposition of the structures of Tmod1 ABS2 (magenta) and Lmod1 ABS2 (green), showing two orientations 90° apart (see ). Note that the structures superimpose well overall, except for the N and C termini (the r.m.s.d. for equivalent Cα is indicated). ( b ) Superimposition of the structures of Tmod1 ABS2 (grey) and TL1 ABS2 (magenta and green, according to ) (see ). ( c ) Superimposition of the structures of complexes of actin (blue) with the hybrid constructs GS1-Tmod1 ABS2 (grey) and GS1-TL1 ABS2 (magenta and green), showing four orientations 90° apart (see ). GS1 is not shown, and actin is only shown for the complex with GS1-TL1 ABS2 . Arrows indicate a slight shift of TL1 ABS2 on actin compared with Tmod1 ABS2 . ( d ) Superimposition of the complexes of actin with TL1 ABS2 (magenta and green) and Tmod1 ABS2 (grey) onto the second actin subunit at the pointed end of the actin filament . Only three subunits of the filament are shown (marine, blue, and grey–purple). ABS2 contacts all three subunits. The arrow indicates a slight shift of TL1 ABS2 compared with Tmod1 ABS2 that inserts it deeper into the groove formed at the interface between actin subunits. ( e ) Same as ( d ) but showing a close-up view of the location of the 11 residues of Tmod1 ABS2 that were mutated to their Lmod1 counterparts in construct Tmod1 ABS2 Mut.

Article Snippet: For expression in mammalian cells, Lmod and Tmod constructs, including Tmod1 N (1–163), Tmod1 ABS2 (164–351), Lmod2 N (1–179), Lmod2 ABS2 (180–367), Lmod1 N (1–298) and Lmod1 ABS2 (299–486), were cloned between the XhoI and BamHI sites of vector pEGFP-N1 (Clontech) or NheI and KpnI sites of vector mCherry2-N1 (Addgene plasmid # 54517).

Techniques: Construct

Journal: Nature Communications

Article Title: How Leiomodin and Tropomodulin use a common fold for different actin assembly functions

doi: 10.1038/ncomms9314

Figure Lengend Snippet: ( a , b ) Cardiomyocytes co-transfected on day 1 with Tmod1 FL -GFP and Tmod1 ABS2 -mCherry (or Tmod1 N -mCherry), fixed 24 h after transfection, and stained with anti-α-actinin antibodies (Z-line marker). Note that Tmod1 FL -GFP and α-actinin are shown separately only in part ( a ) but these markers are also present in part. ( b ). ( c ) Line-scans of each marker (colour coded) along a representative myofibril (insets in parts a , b ). ( d ) Average power spectra resulting from one-dimensional (1D) fast Fourier transform (FFT) analysis of 50 line-scans from six cells transfected with Tmod1 ABS2 (or Tmod1 N ). The frequency of the power peak for Tmod1 FL and α-actinin is 0.572 μm −1 , corresponding to a distance of 1.75 μm between M lines or Z lines, respectively. In contrast, there is no defined power peak in the spectra of Tmod1 ABS2 or Tmod1 N , reflecting a loss of periodicity in their localization. ( e , f ) Cardiomyocytes co-transfected with Lmod2 FL -GFP and Lmod2 ABS2 -mCherry (or Lmod2 FL -mCherry and Lmod2 N -GFP) and stained with anti-α-actinin antibodies. ( g ) Line-scans of each marker (colour coded) along a representative myofibril (insets in parts e , f ). ( h ) Average power spectra resulting from 1D FFT analysis of 50 line-scans from six cells transfected with Lmod2 ABS2 (or Lmod2 N ). All the spectra show similar periodicity, with power peaks at 0.59 and 0.55 μm −1 for Lmod2 ABS2 and Lmod2 N , respectively. The Tmod1 and Lmod2 constructs are defined in Methods and in . Scale bars, 10 μm.

Article Snippet: For expression in mammalian cells, Lmod and Tmod constructs, including Tmod1 N (1–163), Tmod1 ABS2 (164–351), Lmod2 N (1–179), Lmod2 ABS2 (180–367), Lmod1 N (1–298) and Lmod1 ABS2 (299–486), were cloned between the XhoI and BamHI sites of vector pEGFP-N1 (Clontech) or NheI and KpnI sites of vector mCherry2-N1 (Addgene plasmid # 54517).

Techniques: Transfection, Staining, Marker, Construct

Journal: Nature Communications

Article Title: How Leiomodin and Tropomodulin use a common fold for different actin assembly functions

doi: 10.1038/ncomms9314

Figure Lengend Snippet: ( a ) Time course of polymerization and concentration dependence of polymerization rates by Lmod2 FL in the presence of increasing concentrations of Tmod1 FL . Experimental conditions listed on top. The concentration dependence is displayed as the mean of three experiments±s.e.m. ( b ) Time course of polymerization by Lmod2 FL in the presence of Tmod1 FL and TM.

Article Snippet: For expression in mammalian cells, Lmod and Tmod constructs, including Tmod1 N (1–163), Tmod1 ABS2 (164–351), Lmod2 N (1–179), Lmod2 ABS2 (180–367), Lmod1 N (1–298) and Lmod1 ABS2 (299–486), were cloned between the XhoI and BamHI sites of vector pEGFP-N1 (Clontech) or NheI and KpnI sites of vector mCherry2-N1 (Addgene plasmid # 54517).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism

doi: 10.1101/2020.09.18.302901

Figure Lengend Snippet: (A) Schematic representation of CMV 5’ UTR mScarlet reporter (upper panel) and immunofluorescence images of HeLa cells transfected with mScarlet (red) and MBP or MBP-Nsp1 (green), and DNA (blue) (lower panel). (B) Quantification of relative fluorescence intensity of mScarlet in MBP- or MBP-Nsp1-expressing HeLa cells. (C) Gel filtration profiles of MBP-Nsp1 and the MBP-Nsp1–40S complex. Schematic representation of MBP-Nsp1 is shown. Peak positions are labeled. (D) SDS PAGE of MBP-Nsp1 and the MBP-Nsp1–40S complex. (E) A cryo-EM micrograph of the MBP-Nsp1–40S complex.

Article Snippet: SARS-CoV-2 full-length Nsp1 (1-180 aa), Nsp1-NT (1-127 aa) and Nsp1-CT (128-180 aa) were amplified from pDONR207 SARS-CoV-2 NSP1 (Addgene) by PCR and then cloned into pDB-His-MBP or BacMam pCMV-Dest plasmid.

Techniques: Immunofluorescence, Transfection, Fluorescence, Expressing, Filtration, Labeling, SDS Page, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism

doi: 10.1101/2020.09.18.302901

Figure Lengend Snippet: (A) Cryo-EM density of the Nsp1–40S complex. Nsp1 is in cyan. Subunits of 40S that interact with Nsp1 are color-coded. (B) Ribbon diagram of the Nsp1–40S complex. Nsp1 (cyan) binds at the mRNA channel in the cleft between the head and body of the 40S ribosomal subunit. Subunits of 40S that interact with Nsp1 are color-coded. (C) Ribbon diagram and cryo-EM density of SARS-CoV-2 Nsp1 with schematics highlighting its interfaces to 40S. (D) Electrostatic surface representations of Nsp1 binding surfaces to the 40S subunits and rRNA. Buried surface areas are marked. (E) Detailed interactions between Nsp1 and the 40S ribosomal subunit. See also

Article Snippet: SARS-CoV-2 full-length Nsp1 (1-180 aa), Nsp1-NT (1-127 aa) and Nsp1-CT (128-180 aa) were amplified from pDONR207 SARS-CoV-2 NSP1 (Addgene) by PCR and then cloned into pDB-His-MBP or BacMam pCMV-Dest plasmid.

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism

doi: 10.1101/2020.09.18.302901

Figure Lengend Snippet: (A) Workflow of 3D reconstruction. The selected map is boxed in rectangles. (B) Fourier shell correlation (FSC) curves of 3D reconstructed complex of SARS-CoV-2 Nsp1 and human 40S ribosomal subunit.

Article Snippet: SARS-CoV-2 full-length Nsp1 (1-180 aa), Nsp1-NT (1-127 aa) and Nsp1-CT (128-180 aa) were amplified from pDONR207 SARS-CoV-2 NSP1 (Addgene) by PCR and then cloned into pDB-His-MBP or BacMam pCMV-Dest plasmid.

Techniques:

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism

doi: 10.1101/2020.09.18.302901

Figure Lengend Snippet: (A) Summary of Nsp1 constructs and SARS-CoV-2 5’ UTR mScarlet reporter levels. The various Nsp1 constructs are: Nsp1-FL, Nsp1-NT, Nsp1-CT, both Nsp1-NT and Nsp1-CT (Nsp1-NT+CT), Nsp1-linker1, and Nsp1-linker2. (B) Immunofluorescence images of HeLa cells transfected with SARS-CoV-2 5’ UTR mScarlet reporter (red) and MBP, MBP-Nsp1-FL, MBP-Nsp1-NT, MBP-Nsp1-CT, or MBP-Nsp1-N+C (green). (C) Quantification of relative fluorescence intensity of mScarlet in indicated groups from (B) and (E). (D) Quantification of the relative luciferase activity in HEK293T cells transfected with SARS-CoV-2 5’ UTR luciferase reporter and various Nsp1 constructs. Values are means ± S.E.M. obtained from three independent experiments. (E) Immunofluorescence images of HeLa cells transfected with SARS-CoV-2 5’ UTR mScarlet reporter (red) and Nsp1-FL, Nsp1-linker1, or Nsp1-linker2 (green).

Article Snippet: SARS-CoV-2 full-length Nsp1 (1-180 aa), Nsp1-NT (1-127 aa) and Nsp1-CT (128-180 aa) were amplified from pDONR207 SARS-CoV-2 NSP1 (Addgene) by PCR and then cloned into pDB-His-MBP or BacMam pCMV-Dest plasmid.

Techniques: Construct, Immunofluorescence, Transfection, Fluorescence, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism

doi: 10.1101/2020.09.18.302901

Figure Lengend Snippet: (A) Gel filtration profile of the complex between Nsp1-NT (Strep-tagged) and SARS-CoV-2 5’ UTR purified by the Strep-Tactin ® affinity resin from the HeLa cell lysate transfected with the SARS-CoV-2 5’ UTR Nsp1-NT-Strep construct. (B) Anti-Strep tag Western blot (upper panel) and RT-PCR of SARS-CoV-2 5’ UTR (lower panel) of the gel filtration peak fractions in (A). Nsp1-NT co-migrated with SARS-CoV-2 5’ UTR. (C) Construct design of SARS-CoV-2 SL1 5’ UTR and SARS-CoV-2 ΔSL1 5’ UTR-mScarlet reporters (upper panel) and immunofluorescence images of HeLa cells transfected with a mScarlet reporter (red) and MBP or MBP-Nsp1-FL (green) (lower panel) (D) Quantification of data in (C) showing the relative fluorescence intensity of mScarlet in HeLa cells transfected with MBP or MBP-Nsp1-FL. (E) High throughput quantification of mean cellular mScarlet fluorescence intensity when placed downstream of either SARS-CoV-2 5’ UTR, SARS-CoV-2 ΔSL1 5’ UTR, or control 5’UTR, with or without co-expression of MBP-Nsp1. (F) Relative luciferase activity in HEK293T cells after co-transfection of a luciferase reporter and MBP or MBP-Nsp1. The luciferase reporter was placed downstream of SARS-CoV-2 5’ UTR, SARS-CoV-2 SL1 5’ UTR or SARS-CoV-2 ΔSL1 5’ UTR. NC: negative control without adding the luciferase substrate in cells co-transfected with luciferase reporter and MBP. Values are means ± S.E.M. obtained from three independent experiments.

Article Snippet: SARS-CoV-2 full-length Nsp1 (1-180 aa), Nsp1-NT (1-127 aa) and Nsp1-CT (128-180 aa) were amplified from pDONR207 SARS-CoV-2 NSP1 (Addgene) by PCR and then cloned into pDB-His-MBP or BacMam pCMV-Dest plasmid.

Techniques: Filtration, Purification, Transfection, Construct, Strep-tag, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Fluorescence, High Throughput Screening Assay, Control, Expressing, Luciferase, Activity Assay, Cotransfection, Negative Control

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism

doi: 10.1101/2020.09.18.302901

Figure Lengend Snippet: A schematic model depicting the bipartite roles of SARS-CoV-2 Nsp1 during infection. First, Nsp1 blocks host mRNA from binding to the 40S ribosomal subunit due to physical occlusion by the bound Nsp1-CT. Second, Nsp1 supports viral mRNA translation by interacting with SARS-CoV-2 5’ UTR using Nsp1-NT, which results in dissociation of the Nsp1-CT–40S complex to overcome inhibition. This mechanism of evasion of Nsp1-mediated translation inhibition is illustrated by the failure of linker-lengthened Nsp1 to support viral mRNA translation. With the longer linker, the Nsp1-NT–5’ UTR complex can co-exist with the Nsp1-CT–40S complex.

Article Snippet: SARS-CoV-2 full-length Nsp1 (1-180 aa), Nsp1-NT (1-127 aa) and Nsp1-CT (128-180 aa) were amplified from pDONR207 SARS-CoV-2 NSP1 (Addgene) by PCR and then cloned into pDB-His-MBP or BacMam pCMV-Dest plasmid.

Techniques: Infection, Binding Assay, Inhibition