Journal: Cell

Article Title: A trans -complementation system for SARS-CoV-2 recapitulates authentic viral replication without virulence

doi: 10.1016/j.cell.2021.02.044

Figure Lengend Snippet:

Article Snippet: BsaI restriction endonuclease , New England Biolabs , Cat#R3733S.

Techniques: Recombinant, SYBR Green Assay, Electroporation, Gel Extraction, Electron Microscopy, Synthesized, Sequencing, Software

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