Journal: Molecular plant

Article Title: STI PCR: An efficient method for amplification and de novo synthesis of long DNA sequences.

doi: 10.1016/j.molp.2021.12.018

Figure Lengend Snippet: Figure 3. STI PCR enables specific amplification of long genomic DNA fragments. (A–C) (A) PCRs using standard primers and conventional thermo-cycling conditions. The target fragments in (A–C) are indicated by arrows, and the others are non-specific products. The standard (and PS) primers for the 4.9-kb fragment were designed to amplify the Rf4 restorer gene in restorer lines R498 and ZSR5 but not the non-functional rf4 allele in non-restorer lines J23 and 9311. The bands in J23 and 9311 (A and B) therefore represent non- specific products. 9522 and 9311 are japonica and indica rice lines, respectively, which are the same in (B and C). These PCR products (each 3–4 ml) were separated by conventional electrophoresis on 0.8% agarose gel (A–D). M, molecular weight marker l-Hind III. (B) PCRs using standard primers and TIC. (C) PCRs using PS primers and conventional thermo-cycling. (D) STI PCRs using PS primers and TIC. The fragments of 4.9, 10.5, 12.5, and 14.0 kb are the same target sequences used in (A–C). The sizes of all amplified target fragments in (A–D) fit the known sequences in the rice genome (note that DNA fragments of >20 kb could not be effectively separated by conventional agarose gel electrophoresis). The PCRs in (A–D) were performed using KOD FX Neo. (E) Additional one-round STI PCR (using ApexHF HS CL) for amplification of very long genomic sequences from rice, maize, and a human cell line. These PCR products (3–4 ml each) were separated by pulsed-field gel (1.0% agarose) electrophoresis. M, mixed size markers of intact l (48.5 kb) and l-Hind III. (F) Two-round STI PCR for amplification of very long genomic fragments with high specificity. After the first-round STI PCR (using KOD FX Neo), a small amount (0.3–0.5 ml) of the reaction was used as a template for the second PCR using nested specific primers. These can contain an additional 50 tag for cloning or barcoded sequencing. The second-round PCR products were separated by pulsed-field gel electrophoresis. An internal repeat sequence (5.8 or 6.0 kb) that was present in the 38.0- and 38.2-kb target sequences (at the CYP1A1/CYP1A2 locus) of the human cell lines could be efficiently amplified by the nested primers.

Article Snippet: High-fidelity/high-performance DNA polymerase kits of KOD FX Neo (Toyobo, Japan) and ApexHF HS DNA Polymerase CL (Accurate Biotech, Hunan, China), include buffers, were used for PCR tests with an enzyme concentration of 1 unit per 100 ml reaction for <20-kb target sequences (such as 0.4 U in 40 ml reaction/tube) or 1 unit per 90 ml reaction for >20- kb target sequences (such as 0.45 U in a 40 ml reaction/tube).

Techniques: Functional Assay, Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Genomic Sequencing, Cloning, Sequencing, Nucleic Acid Electrophoresis

Journal: Nature Communications

Article Title: A genome-wide CRISPR-Cas9 knockout screen identifies FSP1 as the warfarin-resistant vitamin K reductase

doi: 10.1038/s41467-023-36446-8

Figure Lengend Snippet: a Schematic diagram of genome-wide loss-of-function screening using Brunello lentiviral library with FIXgla-Fas/HEK293 reporter cells. This figure was created with BioRender.com. b Scheme of VKD carboxylation of reporter protein FIXgla-Fas by the vitamin K redox cycle. Apoptosis of reporter cell occurs only when the reporter protein FIXgla-Fas is carboxylated. c Scatterplot of sgRNA enrichment after VKD apoptotic functional screening. Red dots, Fas-associated apoptosis pathway associated proteins; green dots, calcium-dependent proteins; purple dots, GGCX. d Cell-based validation of the candidate genes for vitamin K reduction in the presence of warfarin. Lentivirus containing Cas9 and selected sgRNA were transduced into FIXgla- Met .Luc/HEK293 reporter cells. After puromycin selection for 7 days, survival cells were incubated with 11 µM vitamin K and 5 µM warfarin for 24 h. Carboxylation efficiency of the reporter protein FIXgla- Met .Luc was determined by luminescence ELISA. VKD carboxylation efficiency of non-targeting sgRNA transfected cells was normalized to 100%. e Immunoblotting of HEK293 cells and these cells with their fsp1 gene knocked out (FSP1 KO). Top panel: probed by anti-FSP1 antibody; bottom panel (loading control): probed by anti-GAPDH antibody. Full-length FSP1 is indicated by an arrowhead. f Effect of FSP1 knockout on VKR activity in HEK293 cells. FSP1 was knocked out (-FSP1) from FIXgla-PC/HEK293 or it was re-introduced back into the knockout cells (+FSP1) for VKR activity assay. The carboxylation activity of FSP1 transfected cells (+FSP1) was normalized to 100%. Bottom: Immunoblotting of FSP1 in the corresponding cells using anti-FSP1 as the primary antibody. g HPLC-based conventional VKR in vitro activity assay to determine the reduction of vitamin K to KH 2 . Control: reaction buffer without cell lysate; HEK293, cell lysate of HEK293; HEK293 + FSP1: cell lysate of HEK293 overexpressing FSP1. A same number of HEK293 and HEK293 + FSP1 cells were used for the activity assay. h Warfarin inhibition of FSP1 reducing vitamin K to KH 2 by in vitro activity assay as described above (Fig. 2g). Final concentration of warfarin in the reaction mixture was 100 μM. Data are presented as mean ± SD of three independent experiments ( n = 3) in Fig. 2d, f, h. Similar results were observed at least twice as shown in Fig. 2e, f.

Article Snippet: Mouse anti-GAPDH monoclonal antibody (#60004-1-Ig, diluted 1:6000) and HRP-conjugated anti-His-Tag monoclonal antibody (#HRP-66005, diluted 1:2000) were from Proteintech Group, Inc. Affinity purified sheep anti-human Protein C IgG (#SAPC-AP, diluted 1:4000) and its horseradish peroxidase conjugate (#SAPC-HRP, diluted 1:1000) were from Affinity Biologicals Inc.

Techniques: Genome Wide, Functional Assay, Biomarker Discovery, Selection, Incubation, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Control, Knock-Out, Activity Assay, In Vitro, Inhibition, Concentration Assay

Journal: Nature Communications

Article Title: A genome-wide CRISPR-Cas9 knockout screen identifies FSP1 as the warfarin-resistant vitamin K reductase

doi: 10.1038/s41467-023-36446-8

Figure Lengend Snippet: a Fluorescence confocal microscope imaging of FSP1-sfGFP fusion (green) with cell organelle markers (red) of ER, mitochondrion, and Golgi. FSP1-sfGFP and the cell organelle marker were transiently co-transfected into HEK293 cells for 48 h. b Effect of FSP1 myristylation site mutation (G2A) and mutations of residue responsible for the dinucleotides (NDAH and FAD) binding on VKR activity. Wild-type FSP1 or its mutants was transiently expressed in TKO cells for VKR activity assay. Wild-type FSP1 activity was normalized to 100% (indicated by dotted line). Bottom: Immunoblotting of FSP1, its mutants, and GAPDH (loading control) in the corresponding cells. c AlphaFold model of FSP1 (gray) with FAD (cyan), NADH (gold) and vitamin K (green) based on ligand positions in the yeast NDH-2 enzyme Ndi1 (PDBcode 4G73). The GxGxxG motifs are colored purple (Gly18-Gly23) and orange (Gly149-Gly154) corresponding to interactions with the nucleotide motifs of FAD and NADH respectively. Residues Asp41 and His174 located near the ribose hydroxyls from the two nucleotides are colored pink as is Phe21 from the first GxGxxG region. d FSP1 residues surrounding the isoalloxazine of FAD, nicotinamide of NADH and quinone of vitamin K in the model that were mutated in this study are colored pink. e Effect on FSP1 activity due to mutations surrounding the proposed vitamin K binding site. VKR activity was determined as described above (Fig. 4b). Bottom: Immunoblotting of FSP1, its mutants, and GAPDH (loading control) in the corresponding cells. f Proposed activation mechanism of the activity-based fluorescent probe of vitamin K (VK-ASM) towards FSP1. The probe is inactive until the vitamin K moiety is reduced to hydroquinone, and the rearrangement of the reduced intermediated releases the strong fluorescent tag. g Effect of FSP1 mutations on the reduction of vitamin K and CoQ 10 . Wild-type FSP1 and its mutants were transiently expressed in TKO cells for forty-eight hours, cell lysate was used for activity assay using the activity-based fluorescent probe of vitamin K (VK-ASM) and CoQ 10 (NIR-ASM) as the substrate. Fluorescence intensity of the wild-type FSP1 for each probe was normalized to 100% (indicated by the dotted line). Data are presented as mean ± SD of three independent experiments ( n = 3) in Fig. 4b, e, g. Similar results were observed at least twice as shown in Fig. 4b, e.

Article Snippet: Mouse anti-GAPDH monoclonal antibody (#60004-1-Ig, diluted 1:6000) and HRP-conjugated anti-His-Tag monoclonal antibody (#HRP-66005, diluted 1:2000) were from Proteintech Group, Inc. Affinity purified sheep anti-human Protein C IgG (#SAPC-AP, diluted 1:4000) and its horseradish peroxidase conjugate (#SAPC-HRP, diluted 1:1000) were from Affinity Biologicals Inc.

Techniques: Fluorescence, Microscopy, Imaging, Marker, Transfection, Mutagenesis, Residue, Binding Assay, Activity Assay, Western Blot, Control, Activation Assay