Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: HDX-MS analysis of nsp7, nsp8, and nsp7:nsp8 complexes. Observed percent deuterium uptake of nsp7 (A) and nsp8 (B) during the 1h experiment time course. Secondary structure from PDB: 6YHU annotated in white and predicted secondary structure based on observed percent deuterium in gray.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Overlay of HDX-MS and XL-MS results on nsp7 and nsp8 sequences. Observed inter-nsp7 nsp8 crosslinks only identified in the nsp7:nsp8 1:1 complex are colored in orange and inter-nsp7 nsp8 crosslinks also identified in nsp7:nsp8 1:2 complex are colored in blue. Consolidated change in percent deuterium uptake from and are overlayed on the nsp7 and nsp8 sequences, respectively. Initial Met residue removed from both nsp7 and nsp8 sequences to maintain correct residue numbering.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Overlay of differential HDX-MS perturbation values onto PDB:6YHU. Perturbation values for nsp7 vs nsp7:nsp8 1:1 (A) , nsp8 vs nsp7:nsp8 1:1 (B) , nsp7 vs nsp7:nsp8 1:3 (C) , and nsp8 vs nsp7:nsp8 3:1 (D) colored according to change in percent deuterium levels shown in color bar and respective partner protein colored in pink. Residues not observed by HDX-MS are colored in white.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: XL-MS relative quantification results of nsp7, nsp8, and nsp7:nsp8 complex. (A) Volcano plot summarizing the relative quantification results of crosslinks in nsp8 versus nsp7:nsp8 1:1. Crosslinks enriched in nsp7:nsp8 1:1 complex shown in red box and crosslinks enriched in the nsp8 alone sample shown in blue box. (B) Volcano plot summarizing the relative quantification results of nsp8 versus nsp7:nsp8 1:2 (adjusted) crosslinking reactions. Crosslinks enriched in nsp7:nsp8 1:1 complex shown in red box. Intra-nsp8 crosslinks shown as squares and inter-nsp7 nsp8 crosslinks shown as circles.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Mapping of nsp7 nsp8 inter-protein crosslinks on the dimer (A) and heterotetramer (B) interaction surfaces of the SARS-CoV-2 nsp7 nsp8 heterotetramer crystal structure (PDB: 6YHU). Crosslinks greater than 26 Å distance labeled in red and crosslinks less than 26 Å labeled in blue.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques: Labeling

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Distances of inter-nsp7 nsp8 crosslinks mapped to SARS-CoV-2 nsp7:nsp8 heterotetramer structure (PDB: 6YHU).

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Mapping of nsp7 nsp8 inter-protein crosslinks on the dimer (A), heterotetramer (B), hexadecamer (C) interaction surfaces of the SARS-CoV nsp7 nsp8 hexadecamer cryo-EM structure (PDB: 2AHM). Crosslinks greater than 26 Å distance labeled in red and crosslinks less than 26 Å labeled in blue.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques: Cryo-EM Sample Prep, Labeling

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Distances of inter-nsp7 nsp8 crosslinks mapped to SARS-CoV nsp7:nsp8 hexadecamer structure (PDB: 2AHM).

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Mapping of nsp7-nsp8 inter-protein crosslinks to nsp8-I (A) or nsp8-II (B) of the SARS-CoV-2 replicating polymerase structure (PDB: 6YYT). Crosslinks greater than 26 Å distance labeled in red and crosslinks less than 26 Å labeled in blue.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques: Labeling

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Distances of inter-nsp7 nsp8 crosslinks mapped to SARS-CoV-2 replicating polymerase structure (PDB: 6YYT).

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Distances of inter-nsp7 nsp8 crosslinks mapped to available SARS-CoV-2 replicating polymerase structures composed of nsp7, nsp8, nsp12, and RNA.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

doi: 10.1101/2021.03.06.434214

Figure Lengend Snippet: Distances of inter-nsp7 nsp8 crosslinks mapped to available SARS-CoV-2 replicating polymerase structures composed of nsp7, nsp8, and nsp12.

Article Snippet: Formic acid, trifluoroacetic acid, and UHPLC-grade solvents were purchased from ThermoFisher. pGBW-m4046979 (coding for full-length nsp7, NCBI Reference Sequence: YP_009725303.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145611; http://n2t.net/addgene:145611 ; RRID:Addgene_145611). pGBW-m4046852 (coding for full-length nsp8, NCBI Reference Sequence: YP_009725304.1, codon optimized, with an initial Met and a cleavable C-terminal TEV His 6 tag) was a gift from Ginkgo Bioworks (Addgene plasmid # 145584; http://n2t.net/addgene:145584 ; RRID:Addgene_145584).

Techniques:

Journal: bioRxiv

Article Title: Optimizing multiplex CRISPR/Cas9-based genome editing for wheat

doi: 10.1101/051342

Figure Lengend Snippet: Genome editing using the wheat codon optimized Cas9. (A) Constructs used in the current study. All promoters are shown as white arrows. zUbip-maize ubiquitin gene promoter; OsUbip - rice ubiquitin gene promoter; TaU3p and TaU6p - wheat U3 and U6 promoters, respectively; OsU3p - rice U3 promoter, 2X35Sp - duplicated 35S promoter from the tobacco mosaic virus. Cas9 gene terminator sequences are shown as gray rectangles. Yellow rectangles correspond to the nuclear location signals (NLS), purple rectangles correspond to the target sequence insertion sites in the synthetic single-guide RNA (gRNA), and blue rectangles stand for 3x FLAG sequences. (B) Restriction enzyme digest of a PCR product obtained using the primers flanking the Q gene region targeted by Cas9 and QT1-gRNA (Supplementary Fig. 2). Lane 1 and 6: PCR products from the protoplasts transformed with pA9-GFP, Lanes 2 - 5: PCR products from the protoplasts co-transformed with the gRNA and the different versions of Cas9 from rice (Lane 2), humans (Lane 3), maize (Lane 4), and wheat (Lane 5), respectively. PCR products on Lanes 1 - 5 are digested with the StyI, Lane 6 is non-digested PCR product. Percent of mutations (%) was calculated from the band intensities (see ). (C) Sanger sequencing of the Q gene region targeted by the wheat optimized Cas9 and QT1-gRNA. The wild type sequence is shown on the top row. The target sequence is shown in the red rectangle; the PAM sequence is underlined.

Article Snippet: Wheat codon optimized Cas9 coding sequence (CDS) fused from both sides with the nuclear localization signals (NLS) was synthesized by Integrated DNA Technology (IDT).

Techniques: Construct, Sequencing, Transformation Assay

Journal: bioRxiv

Article Title: Optimizing multiplex CRISPR/Cas9-based genome editing for wheat

doi: 10.1101/051342

Figure Lengend Snippet: Next generation sequencing of barcoded PCR amplicons obtained for multiple Cas9-targeted genomic regions. (A) Workflow of multiplexed PCR amplicon library preparation for NGS. Multiple targets are amplified and barcoded in two rounds of PCR, pooled, and sequenced on the Illumina MiSeq instrument. (B) Illumina reads aligned to the wild type reference sequences. The target sequences are shown in the red rectangles; the PAM sequences are underlined.

Article Snippet: Wheat codon optimized Cas9 coding sequence (CDS) fused from both sides with the nuclear localization signals (NLS) was synthesized by Integrated DNA Technology (IDT).

Techniques: Next-Generation Sequencing, Amplification

Journal: bioRxiv

Article Title: Optimizing multiplex CRISPR/Cas9-based genome editing for wheat

doi: 10.1101/051342

Figure Lengend Snippet: CRISPR/Cas9-mediated multiplex editing in hexaploid wheat using the endogenous tRNA-processing system. (A) The structure of a test construct including the QT1-gRNA (targets the Q gene) flanked by two tRNAs is shown. TaU6p - wheat U6 promoter; QT1 is shown as a green circle; gRNA is shown as a grey rectangle. The gRNAs are released after the tRNA processing of a transcript encoded by a polycistronic gene including the QT1-gRNA and one gRNA without a guide sequence. (B) Alignment of Illumina reads obtained for the QT1-gRNA-targeted regions of the Q gene. The wild type sequence is shown on the top. The QT1 target sequence is shown in the red rectangle; the PAM sequence is underlined. (C) Schematic of tRNA-based processing of a polycistronic gene construct (pBUN421-GLM) containing three tRNA-gRNA blocks. Target sequences GW2T2, LPX1T2, and MLOT1 are shown in purple, blue, and green color, respectively. (D) The NGS of three genomic regions targeted by the pBUN421-GLM construct. The wild type sequence is shown on the top. The target sequences are shown in the red rectangles; the PAM sequences are underlined.

Article Snippet: Wheat codon optimized Cas9 coding sequence (CDS) fused from both sides with the nuclear localization signals (NLS) was synthesized by Integrated DNA Technology (IDT).

Techniques: CRISPR, Multiplex Assay, Construct, Sequencing

Journal: Scientific Reports

Article Title: An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

doi: 10.1038/s41598-021-87068-3

Figure Lengend Snippet: In vitro cleavage assay and OmB cell line gene targeting of IMPA1.1 using Cas9/gRNA RNPs. ( a ) Gene map of Oreochromis IMPA1.1 showing selected gRNA target sites and expanded view of T7 site containing BsrGI restriction site used for analysis of Cas9 cleavage. ( b – e ) Agarose gel images with marker sizes (base pairs or bp) labeled in red ( b and c ) of initial in vitro cleavage assay screen of ten target sites (T1-T10) and reference un-digested substrate amplicon (UD); ( d ) Follow up confirmatory in vitro cleavage assay results of the apparent most efficient cleaving gRNAs from the initial screen showing complete cleavage of substrate amplicon into expected fragments and ( e ) RSM analysis in which the BsrGI digested amplicon from IMPA1.1 T7 CRISPR treated cells (CR D) shows the exact same band pattern as the BsrGI digested wild-type amplicon (WT D) indicating no detectable mutation at the target site. The un-digested amplicons were included for reference (WT UD = wild-type un-digested, CR UD = CRISPR treated un-digested). The gel images in this figure have been significantly cropped to conserve space and increase focus on relevant bands. Full-length gels are presented in Supplementary Figure . Gene map and sequence images were generated using Geneious 11.0.3 (Biomatters, https://www.geneious.com ). Image editing and assembly into complete figures was performed using Inkscape 0.92 ( https://www.inkscape.org ).

Article Snippet: The zebrafish codon optimized Cas9 coding sequence from the pCS2-nCas9n (Addgene # 47929) plasmid and a puromycin resistance gene were cloned downstream of the OmEF1a promoter PCR amplicon separated by a P2A self-cleaving peptide into the base vector between the ITRs to complete the construct.

Techniques: In Vitro, Cleavage Assay, Agarose Gel Electrophoresis, Marker, Labeling, Amplification, CRISPR, Mutagenesis, Sequencing, Generated

Journal: Scientific Reports

Article Title: An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

doi: 10.1038/s41598-021-87068-3

Figure Lengend Snippet: Isolation and selection of Polymerase II promoter for Cas9 expression in transposon plasmid vector. ( a ) PCR isolation of O. mossambicus endogenous promoters illustrated for EF1 alpha. The PCR amplicon spans ~ 150 bp upstream of the transcription start site, exon 1, intron 1, and part of exon 2 to the endogenous start codon. ( b ) Comparison of relative expression strength of candidate promoters for Cas9 quantified by fluorescent intensity of enhanced green fluorescent protein (EGFP). Data shown represent means ± SE (n = 2 per promoter) ( c ) Micrographs showing EGFP expression in OmB cells (green) from SV40, CMV, and OmEF1a EGFP vectors representative of those used for quantitative analysis ( d ) Plasmid map of transposon vector (OmEF1aCas9P2APuroSB) for generation of Cas9 cell line including OmEF1a promoter upstream of Cas9 and puromycin resistance coding sequences (separated by the P2A self-cleaving peptide sequence) in between the two Sleeping Beauty transposon internal terminal repeats (ITRs). Gene and vector maps were generated using Geneious 11.0.3 (Biomatters, https://www.geneious.com ). Bar plot was generated using Rstudio 1.1.456 ( https://rstudio.com ). Image editing and assembly into complete figures was performed using Inkscape 0.92 ( https://www.inkscape.org ).

Article Snippet: The zebrafish codon optimized Cas9 coding sequence from the pCS2-nCas9n (Addgene # 47929) plasmid and a puromycin resistance gene were cloned downstream of the OmEF1a promoter PCR amplicon separated by a P2A self-cleaving peptide into the base vector between the ITRs to complete the construct.

Techniques: Isolation, Selection, Expressing, Plasmid Preparation, Amplification, Comparison, Sequencing, Generated

Journal: Scientific Reports

Article Title: An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

doi: 10.1038/s41598-021-87068-3

Figure Lengend Snippet: Pilot test of Cas9 expressing cells with isolation and validation of clonal Cas9 OmB cell line. ( a and b ) The first trial using TU6 gRNA vector with IMPA1.1 T7 target sequence transfected into puromycin selected Cas9 expressing cells. (a) Schematic of the PCR generated test amplicon used to evaluate mutational efficiency at the IMPA1.1 T7 target including locations of the BsrGI restriction sites and predicted fragment lengths that would result from BsrGI digestion. ( b ) Agarose gels (marker sizes in bp labeled in red) of RSM analysis on IMPA1.1 T7. Left gel consists of un-digested test amplicons from wild-type (WT UD) and T7 CRISPR targeted (CR UD) cells. Right gel consists of BsrGI digested test amplicons from wild-type (WT D) and T7 CRISPR targeted cells (CR D) showing residual fragment length of ~ 437 bp (red arrow) indicative of incomplete digestion of BsrGI restriction site and alteration of the T7 target sequence by Cas9 cleavage. ( c ) Phase contrast image of colony formation of OmEF1aCas9P2ApuroSB vector transfected, puromycin selected OmB cells. ( d and e ) Schematic of primer design for endogenous EF1 alpha control ( d ) and OmEF1a-Cas9 transgene ( e ) verification PCR reactions of presumed Cas9-OmB cell line. ( f and g ) Agarose gels of PCR amplicons (marker sizes in bp labeled in red) verifying genomic integration from genomic DNA template ( f ) and mRNA expression from cDNA template ( g ) of Cas9. Expected EF1 alpha positive control amplicon (C) was observed from both wild-type and presumptive Cas9 OmB cells from both DNA (1007 bp) and cDNA (176 bp) templates. Both of the two expected OmEF1a/Cas9 expression cassette amplicons (1 & 2) were only observed from the Cas9 OmB cells from both DNA (977 bp and 1442 bp) and cDNA (146 bp and 611 bp) templates. The gel images in this figure have been significantly cropped to conserve space and increase focus on relevant bands. Full-length gels are presented in Supplementary Figure . Gene and vector maps were generated using Geneious 11.0.3 (Biomatters, https://www.geneious.com ). Image editing and assembly into complete figures was performed using Inkscape 0.92 ( https://www.inkscape.org ).

Article Snippet: The zebrafish codon optimized Cas9 coding sequence from the pCS2-nCas9n (Addgene # 47929) plasmid and a puromycin resistance gene were cloned downstream of the OmEF1a promoter PCR amplicon separated by a P2A self-cleaving peptide into the base vector between the ITRs to complete the construct.

Techniques: Expressing, Isolation, Biomarker Discovery, Plasmid Preparation, Sequencing, Transfection, Generated, Amplification, Marker, Labeling, CRISPR, Control, Positive Control

Journal: Scientific Reports

Article Title: An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

doi: 10.1038/s41598-021-87068-3

Figure Lengend Snippet: Target sequences, primer pairs used to generate test amplicons by PCR, and sizes of un-digested test amplicons used in RSM analysis of diverse gRNA target testing of the Cas9-OmB1 cell line.

Article Snippet: The zebrafish codon optimized Cas9 coding sequence from the pCS2-nCas9n (Addgene # 47929) plasmid and a puromycin resistance gene were cloned downstream of the OmEF1a promoter PCR amplicon separated by a P2A self-cleaving peptide into the base vector between the ITRs to complete the construct.

Techniques: Sequencing, Amplification

Journal: Scientific Reports

Article Title: An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

doi: 10.1038/s41598-021-87068-3

Figure Lengend Snippet: Mutation analysis from TU6 gRNA expression vector transfected Cas9 OmB cell line treated cells testing multiple targets of different genes. ( a – d ) Agarose gel electrophoresis of RSM analysis of TU6 gRNA expression vector treated Cas9-OmB1 cells targeting diverse loci (marker sizes in bp labeled in red). Except IMPA1.1 T13 and MSTN , nearly all targets showed clear residual undigested amplicon from CRISPR treated cells (CR) relative to equivalent digested amplicon from un-treated OmB cells (WT) indicating at least some successful Cas9 cleavage. For each target, an un-digested control amplicon was included for reference (UD). ( a ) IMPA1.1 targets T7 and T11 shared the same un-digested control (far left lane). ( b ) NFAT5 , the two targets regions were amplified by different primer pairs and thus have separate un-digested controls. ( c ) Presumed non-essential gene control targets. Clear residual un-digested amplicon from NANOS3 target but lack of conclusive results from MSTN target due to low quantity of DNA. ( d ) The NR3C1 targets T1 and T2 shared the same un-digested control (far left lane). ( e ) TIDE indel analysis of amplicons from TU6 vector treated Cas9-OmB cells and from previous IMPA1.1 targeting Cas9/gRNA RNP treated cells. Note the common target between the two approaches ( IMPA1.1 T7), 14.1% from the vector versus 0.9% from the RNP methods. Bar plot was generated using Rstudio 1.1.456 ( https://rstudio.com ). Image editing and assembly into complete figures was performed using Inkscape 0.92 ( https://www.inkscape.org ).

Article Snippet: The zebrafish codon optimized Cas9 coding sequence from the pCS2-nCas9n (Addgene # 47929) plasmid and a puromycin resistance gene were cloned downstream of the OmEF1a promoter PCR amplicon separated by a P2A self-cleaving peptide into the base vector between the ITRs to complete the construct.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Transfection, Agarose Gel Electrophoresis, Marker, Labeling, Amplification, CRISPR, Control, Generated

Journal: Scientific Reports

Article Title: An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

doi: 10.1038/s41598-021-87068-3

Figure Lengend Snippet: CRISPR/Cas9 editing confirmation and efficiency comparisons of different U6 promoters by analysis of NANOS3 gRNA target. ( a and b ) Sequencing results of individual alleles from plasmid sub-cloned test amplicons. ( a ) For the NANOS3 target, 17 out of 21 amplicons (81%) were altered at the target site (4 wild-type sequences not shown). ( b ) For the NFAT5 T10 target, 3 out of 19 amplicons (16%) were altered at the target site. The remaining 16 sequences were identical to the O. niloticus reference sequence except for an extra nucleotide (an A when reading the gene sequence 5′ to 3′, a T when reading the gRNA sequence 5′ to 3′) 5 base pairs from the PAM sequence (highlighted in green). ( c ) Alternate cloning strategy for changing gRNA target sequence in expression vector illustrated. Utilizes a mutated TU6 (TU6m) in which a single nucleotide was changed adjacent to the TSS generating a ClaI restriction site. The TU6m is included in the base vector in which new gRNA target sequences can be added by annealed oligos. ( d ) Mutational efficiency quantified by TIDE indel% analysis of four different U6 promoters using the same gRNA target ( NANOS3 ) showing superior editing obtained from both versions of the tilapia U6 promoters (over fivefold over all others). The Human and Zebrafish U6 promoters were not statistically significant from the no U6 control. Gene maps and DNA sequence images were generated using Geneious 11.0.3 (Biomatters, https://www.geneious.com ). Bar plot was generated using Rstudio version 1.1.456 ( https://rstudio.com ). Image editing and assembly into complete figures was performed using Inkscape version 0.92 ( https://www.inkscape.org ).

Article Snippet: The zebrafish codon optimized Cas9 coding sequence from the pCS2-nCas9n (Addgene # 47929) plasmid and a puromycin resistance gene were cloned downstream of the OmEF1a promoter PCR amplicon separated by a P2A self-cleaving peptide into the base vector between the ITRs to complete the construct.

Techniques: CRISPR, Sequencing, Plasmid Preparation, Clone Assay, Cloning, Expressing, Control, Generated