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: 21 The FnCas12a and AsCas12a expression constructs were synthesized by GenScript.
Techniques: Transformation Assay, Construct, Plasmid Preparation, Homologous Recombination, Expressing, Marker, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Selection, Sequencing