SOFTWARE

SHEWANELLA TRANSPOSASE CALCULATOR

Github repository : https://github.com/keitzlab/ShewCASTgRNAs
CAST Publication : https://www.nature.com/articles/s41467-024-50816-w
Shew CAST gRNA Calculator: https://shewcastgrna.streamlit.app (developed by Ismar Miniel Mahfoud)

The Shewanella CAST gRNA Generator is a computational tool for use in the design, construction, and application of CRISPR-associated transposons (CASTs) for genome modification in S.oneidensis and other microbial species. In short, gRNAs can be used to guide the insertion of transposable elements to specific sequences in the target genome. The pR6K-GFP-CASTIF plasmid (Addgene catalog # 199653) contains a constitutively expressed GFP gene flanked by BsaI Type IIS cut sites as well as a chloramphenicol resistance gene to be inserted as the transposon cargo. 

After selection of the target gene from Shewanella genes available in the KEGG database, the program generates three gBlock DNA sequences containing a 20 bp gRNA sequence flanked by the appropriate repeat sequences as well as the BsaI cut sites designed to complement those on the pR6K-GFP-CASTIF plasmid. The GFP dropout plasmid allows for quick screening of transformants into MFDpir or WM3064 strains of E.coli used for conjugation of the plasmid into Shewanella. The app includes instructions for cloning the gRNA into the plasmid and links to a Shewanella conjugation protocol. Users are also able to manually enter DNA sequences to generate gRNAs for non-Shewanella genes.