An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

Mycoplasma bovis is a minimal bacterium infecting cattle, which causes a wide variety of symptoms and is impacting dairy and beef producers worldwide. Part of the difficulty in research surrounding M. bovis, and other mycoplasmas, is the lack of efficient genome editing tools. We previously presented a proof of concept of a transposon-based CRISPR-Base Editor system to introduce targeted mutations in M. bovis. In this work, we present several improvements of the existing tool: multi-loci targeting through addition of a second guide RNA; increased number of targetable loci by using an engineered Cas9 with AT-rich PAM specificity, and elimination of the CRISPR-Base Editor from the generated mutant through either transposon excision or use of an oriC plasmid. We also propose a dedicated bioinformatic tool to identify target sequences in genes of a given genome. We applied this software to demonstrate the potential of our improved tools in M. bovis and other mycoplasma of veterinary and human interest that currently lack genome editing methods.