Journal: bioRxiv

Article Title: Synthetic circRNAs employ IRES activity for translation in cells and in cell-free translation systems

doi: 10.64898/2026.03.28.715045

Figure Lengend Snippet: (A) Experimental outline of the in vitro generated circRNA-based reporter assay based on the mRuby3_circ_EGFP_ScaI_v3-4 plasmid (pKL480) for evaluation of IRES activity of different IRES inserts, including inverse sequences as controls for circRNA translation dependent on insert length and GC-content. Following DNA template synthesis (PCR amplification from plasmid backbone using poly(A)- and poly(T)-including primers), circRNA was generated by T7 in vitro transcription. Linear RNA species were removed by RNase R-treatment and column purification. Resulting circRNA was subsequently used for in-cell transfection or for in vitro translation systems. EGFP and 3xHA-Nluc reporter systems were used throughout the following analyses. The plasmid-encoded mRuby was not used. (B) Illustration of the circularization reaction mediated by group I dt introns leading to self-spliced circRNA. The observed sequence scar formed by the remaining parts of the td introns are highlighted in light and dark red. Remaining split introns were generated as side products and need to be removed. (C) Overview of the tested IRES sequences indicating length and viral or cellular origin of the IRESes. (D) Quality control of the generated EGFP reporter including circRNAs using 1% FA-agarose gel. Linear side products (upper band) disappear by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). 500 ng total RNA was loaded per lane. The RiboRuler High Range RNA Ladder (Thermo, #SM1821) serves as a reference. (E) Quality control of the generated 3xHA-Nluc encoding circRNAs using 1% FA-agarose gel. Linear side products (upper band) are mainly degraded by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). Only Dlx1 shows less efficient circularization leading to a higher concentration of side products. Therefore, the RNase R incubation time was increased to 90 min. 500 ng total RNA was loaded per lane. RNA species: linear (L), circular (C), introns (I). (F) Quality control, using the High Sensitivity RNA ScreenTape, of the generated circRNA before purification (upper panel) and after RNase R digestion (lower panel). Remaining contaminants can be observed as light grey bands.

Article Snippet: The PCR products were purified using the Monarch PCR & DNA Cleanup kit (NEB, T1030L) and 1 μg DNA was subsequently used for in vitro transcription using the High Scribe T7 High Yield RNA synthesis Kit (NEB, E2040S) according to manufacturer’s protocol (20 μL total reaction volume) supplemented with 40 U RiboLock RNase inhibitor (Thermo, EO0381) and incubated for 2 hrs at 37°C and 800 rpm.

Techniques: In Vitro, Generated, Reporter Assay, Plasmid Preparation, Activity Assay, Amplification, Purification, Transfection, Sequencing, Control, Agarose Gel Electrophoresis, Concentration Assay, Incubation

Journal: Cureus

Article Title: Evaluation of Fibroblast Growth Factor Receptor 3 (FGFR3) and Tumor Protein P53 (TP53) as Independent Prognostic Biomarkers in High-Grade Non-muscle Invasive Bladder Cancer

doi: 10.7759/cureus.65816

Figure Lengend Snippet: List of forward and reverse primers used in the methylation-specific PCR study for each gene.

Article Snippet: Methylation-specific polymerase chain reaction (MSP) EZ DNA methylation gold kit (Cat no. # D5005; Zymo Research, Irvine, CA) was used for the bisulfite conversion of DNA.

Techniques: Methylation, Sequencing

Journal: The CRISPR Journal

Article Title: Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles

doi: 10.1089/crispr.2020.0086

Figure Lengend Snippet: Activity of Cas12a orthologs at varied temperatures. (A) Cas12a/SpCas9 proteins with their respective crRNA/sgRNA and buffer (same as indicated in (B)) were combined to form the ribonucleoprotein (RNPs) for 5 min at the temperatures indicated. The double-stranded DNA (dsDNA) cleavage reactions were initiated by adding the 5′-FAM-labeled dsDNA containing the target site preincubated at the designated incubation temperatures. Digested fragments were resolved by capillary electrophoresis, and peaks corresponding to cleaved and intact substrates were quantified. Data are shown as the mean ± standard deviation (SD) of three experimental replicates. (B) Recombinant crRNA-free Cas proteins or crRNA-loaded RNPs were subjected to thermal melting analysis using Nano differential scanning fluorimetry (Supplementary Figure S1). The melting temperature (Tm) of three experimental replicates are shown, with means ± SD indicated.

Article Snippet: PCR products were purified using a Monarch PCR and DNA Cleanup Kit (NEB #T1030S). crRNA for Cas12a was synthesized (NEB3), and the guide for Cas9 was generated using the EnGen sgRNA synthesis kit S. pyogenes (NEB #E3322S) using the template for top strand (NEB4).

Techniques: Activity Assay, Labeling, Incubation, Electrophoresis, Standard Deviation, Recombinant, Nano Differential Scanning Fluorimetry

Journal: The CRISPR Journal

Article Title: Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles

doi: 10.1089/crispr.2020.0086

Figure Lengend Snippet: Transformation of HR-FnCas12a mediated editing plasmids in B. smithii ET 138. (A) Schematic representation of the pFnCas12a_Δgene-of-interest (goi)-HR construct. The fncas12a gene was introduced to the pNW33n vector backbone. Homologous recombination (HR) flanks were introduced upstream fncas12a gene and encompassed the 1 kb upstream and 1 kb downstream region of the goi in the B. smithii genome. A crRNA-expressing module was introduced downstream the fncas12a gene. Other elements on the plasmid are origin of replication (ori), replication protein (repB), and chloramphenicol-resistance marker (CmR). (B) Sequential transfer scheme of B. smithii cultures to evaluate FnCas12a editing efficiency; detailed description of the protocol can be found in the Methods section. (C) Agarose gel electrophoresis showing the results from polymerase chain reaction on the genomic DNA of B. smithii cultures transformed with pFnCas12a_ΔpyrF-HR_Sp1 (1), pFnCas12a_ΔpyrF-HR_Sp2 (2), and pFnCas12a_ΔpyrF-HR_NT (NT) in two different selection media (TVMYxgu and LB2xgu). The last two lanes are the negative (wild type) and positive (ΔpyrF) controls that correspond to DNA fragments 2.9 and 2.2 kb long, respectively. (D) Representative image of the sequence verification of the desired pyrF gene deletion by Sanger sequencing.

Article Snippet: PCR products were purified using a Monarch PCR and DNA Cleanup Kit (NEB #T1030S). crRNA for Cas12a was synthesized (NEB3), and the guide for Cas9 was generated using the EnGen sgRNA synthesis kit S. pyogenes (NEB #E3322S) using the template for top strand (NEB4).

Techniques: Transformation Assay, Construct, Plasmid Preparation, Homologous Recombination, Expressing, Marker, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Selection, Sequencing

Journal: bioRxiv

Article Title: A Generalized Platform for Artificial Intelligence-powered Autonomous Protein Engineering

doi: 10.1101/2025.02.12.637932

Figure Lengend Snippet: The site-directed mutagenesis protocol used a 27 bp mutagenic primer (12+3+12) for generating mutations in the ORF. Each mutation site required two mutagenic primers to amplify the adjacent fragments. Another set of primers were used for PCR of vector backbone. The three PCR fragments contained overlapping regions of 27–40 bp. The template plasmids for PCRs were linearized with appropriate restriction enzyme. For ORF PCR, ∼200 pg of template DNA was used per reaction. The PCR reactions were treated with Dpn I to eliminate the original templates, followed by HiFi assembly of the backbone and two adjacent ORF fragments.

Article Snippet: For PCR templates, the plasmids were linearized with restriction enzymes and purified using a PCR cleanup kit (Zymo #D4018).

Techniques: Mutagenesis, Plasmid Preparation

Journal: bioRxiv

Article Title: A Generalized Platform for Artificial Intelligence-powered Autonomous Protein Engineering

doi: 10.1101/2025.02.12.637932

Figure Lengend Snippet: Module 1 of the workflow prepared mutagenesis PCR for 96 mutants in a 96-well PCR plate. The templates and primers for PCR were mixed using worklists on the Tecan Fluent and Echo liquid handler. PCR success was measured by adding 2.5 μL of the PCR reaction to 47.5 μL of 1x Evagreen dye (Biotium #31000) and measuring fluorescence ( λ ex = 498 nm / λ em = 535 nm) with the Tecan Infinite plate reader. 25 μL PCR product was transferred to a new PCR plate, and 1 μL of Dpn I (NEB #R0176) was added, followed by incubation at 37 °C. The Dpn I-treated PCR products were then transferred to a 384-well plate, along with the vector backbone, and a worklist guided the mixing of the correct fragments in the Echo liquid handler for HiFi assembly. After a 30-minute HiFi assembly at 50 °C, competent DH5α cells in a 96-well plate were transformed by heat shock on the Tecan Fluent using onboard heating/cooling blocks. The cells were plated on 8-well omnitray agar plates containing LB + 50 μg/mL kanamycin and incubated overnight at 37 °C in a Cytomat automated shaking incubator.

Article Snippet: For PCR templates, the plasmids were linearized with restriction enzymes and purified using a PCR cleanup kit (Zymo #D4018).

Techniques: Mutagenesis, Fluorescence, Incubation, Plasmid Preparation, Transformation Assay

Journal: Cell reports

Article Title: Nanoparticle-based itaconate treatment recapitulates low-cholesterol/low-fat diet-induced atherosclerotic plaque resolution

doi: 10.1016/j.celrep.2024.114911

Figure Lengend Snippet:

Article Snippet: Direct-zol RNA Miniprep Kit , Zymo Research , R2054.

Techniques: Purification, Plasmid Preparation, Produced, Recombinant, Concentration Assay, Saline, Labeling, Membrane, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Phospholipid Assay, BIA-KA, RNAscope, HD Assay, Polymer, Sequencing, Expressing, Software, Microscopy