vector backbone px330  (Addgene inc)

Journal: Molecular Therapy. Nucleic Acids

Article Title: MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

doi: 10.1016/j.omtn.2022.12.020

Figure Lengend Snippet: Establishing an efficient transfection system and determining the cleavage efficiencies of MSTN and FGF5 sgRNAs in SFFs (A) Fluorescence expression in SFFs transfected with the pEGFP-N1 plasmid by the indicated methods. (B) Determination of the efficiencies of lipofection and nucleofection by flow cytometry at 48 h post transfection. (C and D) Schematic of sgRNAs specific to exon 3 of the sheep MSTN and FGF5 loci. The CRISPR RNA (crRNA) sequences are indicated in red typeface and the PAM in blue. Primers #1 and #2 were designed for PCR amplification of the region around the targeted sites to detect the indel frequencies of MSTN and FGF5 genes, respectively. (E and F) TA cloning and Sanger sequencing analysis of the mutation types and efficiencies detected in the pX330-MSTN or pX330-FGF5 plasmid-transfected SFFs. Deletions are indicated by a dashed line (−), and newly added nucleotides are highlighted in green. The colony numbers are surrounded by brackets.

Article Snippet: The Cas9 and U6-sgRNA co-expression vector backbones pX330 and pX458 were purchased from Addgene (plasmid ID: 42230 and 48138).

Techniques: Transfection, Fluorescence, Expressing, Plasmid Preparation, Flow Cytometry, CRISPR, Amplification, TA Cloning, Sequencing, Mutagenesis

Journal: Molecular Therapy. Nucleic Acids

Article Title: MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

doi: 10.1016/j.omtn.2022.12.020

Figure Lengend Snippet: Optimizing conditions and identifying potential enhancers for efficient HDR in SFFs (A) Schematic of the targeting strategy for knockin T2A-EGFP with HA in the length range of 50–1,000 bp to the sheep MSTN locus. The T2A-EGFP repair dsDNA templates were amplified from the MSTN donor plasmid by PCR. (B) Sanger sequencing of the 5′ and 3′ junction regions (T2A-EGFP) upon correct targeting at the MSTN locus. The intentionally added cytosine (C) is marked in gray, and the termination codon (TAA) of EGFP gene is marked in brown. (C) FACS analysis of EGFP-positive cells at 48 h after transfection of the pX330-MSTN plasmid and MSTN-T2A-EGFP with 50, 100, 300, 500, 750, or 1,000 bp HA repair template into SFFs. A representative FACS result of each length is shown. The cells transfected with the pX330-MSTN plasmid alone, or with MSTN-T2A-EGFP with the 0 bp HA repair template alone, were used as a negative control. (D) Effect of the HA length on HDR efficiency at the MSTN locus in SFFs. (E) FACS analysis of EGFP-positive cells at 48 h after transfection of the pX330-MSTN plasmid and different amounts of MSTN-T2A-EGFP with the 500 bp HA repair template into SFFs. A representative FACS result of each amount is shown. The cells transfected with the pX330-MSTN plasmid alone, or with MSTN-T2A-EGFP with the 500 bp HA repair template (1 pmol) alone, were used as a negative control. (F) Effect of the amount of repair template on HDR efficiency at the MSTN locus in SFFs. (G) FACS analysis of EGFP-positive cells at 48 h after transfection of the pX330-MSTN plasmid and MSTN-T2A-EGFP with 1,000 bp HA (6 pmol) into SFFs. Representative HDR efficiency of samples treated with 0.1% DMSO, 20 μM nicotinamide, 1.4 mM Ca 2+ , 5 ng/mL IL-10, 10 μM pifithrin-β, 0.2 μM SF2523, 10 μM SCR7, 0.2 μM CC-115, and 0.05 μM PIK-75 are shown. DMSO-treated cells were used as a control. pX330-MSTN plasmid-transfected cells and MSTN-T2A-EGFP with the 1,000 bp HA repair template-transfected cells were used as negative control. (H) Effects of varying concentrations of different potential enhancers on HDR efficiency in SFFs. n = 3 biological replicates. Error bars represent SD. Significance was calculated using Tukey’s multiple comparison test or Student’s t test: ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: The Cas9 and U6-sgRNA co-expression vector backbones pX330 and pX458 were purchased from Addgene (plasmid ID: 42230 and 48138).

Techniques: Knock-In, Amplification, Plasmid Preparation, Sequencing, Transfection, Negative Control, Control, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

doi: 10.1016/j.omtn.2022.12.020

Figure Lengend Snippet: MDM2 antagonists promote CRISPR-Cas9-mediated precise genome editing in SFFs (A) Schematic of the targeting strategy for knockin T2A-EGFP with 1,000 bp HA to the sheep FGF5 locus. (B) Detection of the relative mRNA expression levels of the p21 and Cdc25C genes in the Cas9/MSTN sgRNA/HDR/MDM2 antagonist-treated cells at 24 h post transfection. (C and E) FACS analysis of EGFP-positive cells at 48 h after transfection of the pX330-MSTN/FGF5 plasmid and MSTN/FGF5-T2A-EGFP repair template with 1,000 bp HA into SFFs. Representative FACS results of samples treated with 0.1% DMSO, 10 μM pifithrin-β, 10 nM RITA, 10 μM Nutlin3, 1.5 μM CTX1 alone, or the MDM2 antagonist mixture are shown. (D and F) Effects of p53 inhibitor and MDM2 antagonists on HDR efficiency at the MSTN and FGF5 loci. DMSO-treated transfected cells were used as a control. n = 3 biological replicates. Error bars represent SD. Significance was calculated using Student’s t test: ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: The Cas9 and U6-sgRNA co-expression vector backbones pX330 and pX458 were purchased from Addgene (plasmid ID: 42230 and 48138).

Techniques: CRISPR, Knock-In, Expressing, Transfection, Plasmid Preparation, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

doi: 10.1016/j.omtn.2022.12.020

Figure Lengend Snippet: MDM2 antagonists promote CRISPR-Cas9-mediated generation of knockin SFF lines (A) Schematic of the targeting strategy for knockin CMV-mCherry-pA with 1,000 bp HA to the sheep MSTN locus. The insertion of the exogenous donor template was detected by PCR (primer #3), and the size of the amplified band was 1,342 bp. Primer #3F was an internal forward primer in the SV40 poly(A) signal, while #3R was designed outside of the flanking arm. (B) Summary of generation of mCherry knockin SFF lines via the CRISPR-Cas9 system. After 48 h of small-molecules treatments, the cells transfected with the pX330-MSTN plasmid and CMV-mCherry-pA with the 1,000 bp HA donor template were singly picked into 96-well plates. After 15 days of cell culture, each of the mCherry-positive single-cell colonies were partly lysed and then identified the occurrence of correct integration by PCR. (C) Genotyping results of the mCherry-positive single-cell colonies using primer #3 (+, mCherry knockin; −, wild type). Lanes P and N served as positive (genomic DNA was extracted from the pX330-MSTN plasmid and CMV-mCherry-pA with the 1,000 bp HA donor template-transfected SFFs) and negative (genomic DNA was extracted from wild-type SFFs) controls, respectively. (D) Fluorescence expression in mCherry knockin and mCherry-negative SFF lines. Significance was calculated using χ 2 test: ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: The Cas9 and U6-sgRNA co-expression vector backbones pX330 and pX458 were purchased from Addgene (plasmid ID: 42230 and 48138).

Techniques: CRISPR, Knock-In, Amplification, Transfection, Plasmid Preparation, Cell Culture, Fluorescence, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

doi: 10.1016/j.omtn.2022.12.020

Figure Lengend Snippet: Effect of cell-cycle stage on HDR efficiency in SFFs (A and B) Cell-cycle distribution of co-transfected SFFs after treatment with DMSO, 10 μM pifithrin-β, 10 nM RITA, 10 μM Nutlin3, and 1.5 μM CTX1 for 24 h. Samples were stained by PI and analyzed by FACS. (C and D) Analysis of SFFs with different cell-cycle blocks by PI staining and flow cytometry. (E) FACS analysis of the HDR efficiency upon transfection with the pX330-MSTN plasmid and MSTN-T2A-EGFP with the 1,000 bp HA repair template into SFFs in different phases of the cell cycle. (F) Effects of different cell-cycle blocks on HDR efficiency. DMSO-treated cells served as a control. n = 3 biological replicates. Error bars represent SD. Significance was calculated using Student’s t test: ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: The Cas9 and U6-sgRNA co-expression vector backbones pX330 and pX458 were purchased from Addgene (plasmid ID: 42230 and 48138).

Techniques: Transfection, Staining, Flow Cytometry, Plasmid Preparation, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

doi: 10.1016/j.omtn.2022.12.020

Figure Lengend Snippet: p53 mutation suppresses CRISPR-Cas9-mediated precise genome editing in SFFs (A) Schematic diagram of the p53 gene, with mutation in exons 1 and 3. The crRNA sequences are indicated in red typeface and the PAM in blue. The indels in the p53 locus were detected by PCR (primer #4), and the predicted product sizes are 1,055 (wild type [WT]) and 562 bp (double knockout [dKO]). Primer #5 was designed for reverse transcription PCR (RT-PCR) to detect the mutation of p53 mRNA in SFFs. The predicted amplicon sizes are shown at the right side. (B) Summary of generation of p53 knockout SFF lines using the CRISPR-Cas9 system. sgRNA1 and sgRNA2 target exons 1 and 3 of the p53 gene, respectively. (C) Genotyping results of p53 gene knockout single-cell colonies selected in the sgRNA1 + sgRNA2-induced gene targeting using primer #4 (−, KO allele; I, whether this allele is mutated needs further verification by sequencing). Colonies with gene KO in at least one allele are marked in blue (I/−), and the biallelic mutation colonies are marked in red (−/−: C7, C16, and C23). (D) Sanger sequencing analysis of the three screened p53 −/− colonies. Nucleotide deletions are indicated by a dashed line (−). (E) RT-PCR results of the WT, C7, C16, and C23 cell lines using primer #5. (F and G) Determination of the HDR efficiency by flow cytometry at the MSTN locus in p53 +/+ and p53 −/− SFFs at 48 h after transfection of the pX330-MSTN plasmid and MSTN-T2A-EGFP with the 1,000 bp HA repair template. The MSTN donor-transfected cells were used as a negative control. (H and I) Determination of the HDR efficiency by flow cytometry at the FGF5 locus in p53 +/+ and p53 −/− SFFs at 48 h after transfection of the pX330-FGF5 plasmid and FGF5-T2A-EGFP with the 1,000 bp HA repair template. The FGF5 donor-transfected cells were used as a negative control. (J and K) Detection of the relative mRNA expression levels of the key regulatory genes involved in the HDR pathway at 24 h after transfection of the pX330-MSTN plasmid and MSTN-T2A-EGFP with the 1,000 bp HA repair template in p53 +/+ and p53 −/− SFFs. The data were normalized to β-actin, and p53 −/− SFFs were used as a control. n = 3 biological replicates. Error bars represent SD. Significance was calculated using Student’s t test: ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: The Cas9 and U6-sgRNA co-expression vector backbones pX330 and pX458 were purchased from Addgene (plasmid ID: 42230 and 48138).

Techniques: Mutagenesis, CRISPR, Double Knockout, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Amplification, Knock-Out, Gene Knockout, Sequencing, Flow Cytometry, Transfection, Plasmid Preparation, Negative Control, Expressing, Control