human codon optimized cas13a expression plasmid  (Addgene inc)

Journal: Molecular Medicine

Article Title: The SARS-CoV-2 nucleocapsid protein induces microglia senescence-mediated cognitive impairment via Glycolysis

doi: 10.1186/s10020-025-01410-3

Figure Lengend Snippet: SARS-CoV-2 N protein drives the senescence of BV2 microglial cells by triggering mitochondrial dysfunction. BV2 microglial cells were treated with Mdivi-1 (100 nM) 30 min before Codon-optimized pLJM1-SARS-CoV-2 N-FLAG transfection. A Representative image of BV2 cells loaded with the mitochondrial membrane potential indicator JC-1 (bar = 20 μm). B Mitochondrial morphology stained with TOM20 in BV2 cells (bar = 2 μm). C - D The expression levels of p-DRP1 and DRP1 proteins in BV2 microglial cells detected by Western blot ( n = 3). Bar graph data represent the mean ± SD of the target protein intensity (normalized to DRP1) in the experimental group relative to that in the control group. E Representative images of BV2 cells loaded with the mitochondrial membrane potential indicator JC-1 (bar = 20 μm). F The expression levels of p19 , p21 , p53 , Il-1β , and Tnf-α mRNA in BV2 microglial cells detected by real-time PCR ( n = 3). G SA-β-gal staining was performed after transfection of Codon-optimized pLJM1-SARS-CoV-2 N-FLAG and treatment with Mdivi-1 for 36 h (bar = 10 μm). H The fluorescence intensity of Ki67 (green) was detected by immunofluorescence (bar = 50 μm). I - J The expression levels of p-DRP1, p53, p21, p16, and γ-H2AX proteins in BV2 microglial cells were detected by Western blot ( n = 3). Bar graph data represent the mean ± SD of the target protein intensity (normalized to α-tubulin) in the experimental group relative to that in the control group. Comparisons between the two groups were made with an unpaired t -test. Differences among multiple groups were performed using ANOVA. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Codon-optimized pLJM1-SARS-CoV-2 N-FLAG was cloned from SinoBiological (cat# VG40588-NF) using BamHI and EcoRI.

Techniques: Transfection, Membrane, Staining, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Fluorescence, Immunofluorescence

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Engineering LwaCas13a to enhance RNA knockdown efficiency (A) Schematic diagram of the mammalian dual-fluorescence reporter system. (B) Sequence alignment of LwaCas13a and its homologous proteins. Candidate mutagenic regions are marked in the red box. (C) Comparison of the cleavage effect of LwaCas13a mutant and WT in HEK293T cells. The fluorescence intensity level of EGFP represents the targeted cleavage effect of Cas13a (left); the fluorescence intensity level of mCherry represents the collateral cleavage effect of Cas13a (right). (D) Structure-guided mutation and corresponding activity assessment. Mutation candidates in the HEPN1-II domain are marked in red lines. Data were compared using a two-tailed unpaired Mann-Whitney U test. Data are presented as median ± IQR ( n = 4). ∗ p < 0.05; ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: Knockdown, Fluorescence, Sequencing, Comparison, Mutagenesis, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Activity profiling of Cas13a mutations (A) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a double mutants. (B) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a triple mutants. (C) The targeted (EGFP) and collateral (mCherry) cleavage activities of all the Cas13a beneficial mutants. Data were compared using a two-tailed unpaired Mann-Whitney U test. Data are presented as median ± IQR ( n = 4). ∗∗∗ p < 0.001.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Screening and performance of crRNA mutants (A) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a combined with crRNA mutants with 3' end C 3 N extensions. (B) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a combined with crRNA mutants with 3' end U 3 V (A/C/G) or U 4 V (A/C/G) extensions. (C) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a combined with crRNA mutants with 5' end N 4 extensions. (D) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a combined with crRNA mutants with both 5' and 3' end beneficial extensions. (E) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a combined with all the beneficial crRNA mutants. Data were analyzed using the Kruskal-Wallis test followed by Dunn’s multiple comparisons test, comparing each group to the non-targeting (NT) crRNA control. Data are presented as median ± IQR ( n = 4). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Cytotoxicity evaluation of CRISPR-Cas13a system with engineered crRNAs (A) Cell viability was determined using CCK-8 reagent at 24, 48, 72, and 96 h post-transfection. The line chart is the overall graph. WT, wild-type LwaCas13a; NT-crRNA, nontarget crRNA; T-crRNA, target crRNA. (B) Bar charts show specific data for each time point. Data were compared using a two-tailed unpaired Mann-Whitney U test. Data are presented as median ± IQR ( n = 3). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: CRISPR, CCK-8 Assay, Transfection, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Engineered CRISPR-Cas13a system exhibits superior cleavage activity in mammalian cells (A) Cleavage activity was determined by dual fluorescence system in HEK293T cells. NT, nontarget crRNA; T, target crRNA ( n = 4). (B) Knockdown efficiency of six endogenous transcripts was determined by qPCR ( n = 3). (C and D) Antiviral assays targeting MuV HN (left) and P (right) genes. Viral titers in Vero-E6 cell supernatants were determined by qPCR ( n = 3). Data were compared using a two-tailed unpaired Mann-Whitney U test. Data are presented as median ± IQR. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: CRISPR, Activity Assay, Fluorescence, Knockdown, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Engineered Cas13a mutant and crRNA mutants contribute to enhancing Cas-crRNA interactions (A) BLI kinetic analysis of the interaction between wild-type/mutant Cas13a and wild-type/mutant crRNAs. Kinetic parameters were from a 1:1 binding model. (B) Comparison of the KD values in graph A. Data were compared using a two-tailed unpaired Mann-Whitney U test. Data are presented as median ± IQR ( n = 4). ∗ p < 0.05; ns, not significant.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: Mutagenesis, Binding Assay, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Structural modeling of wild-type and engineered CRISPR-Cas13a system (A) Structural diagram of the binary complex between wild-type/mutant Cas13a and wild-type crRNA, along with a local magnification. (B) Structural diagram of the binary complex between mutant Cas13a and M3crRNA, along with a local magnification. Mutant amino acid residues are labeled in rose; amino acid residues in contact with the 34th to 42nd bases of crRNA are labeled in pink.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: CRISPR, Mutagenesis, Labeling