RNA-Guided Microbial Genome Editing Using Group II Introns and CRISPR-Cas Systems

Microbial genome engineering presents several challenges. Many prokaryotic organisms are not amenable to homologous recombination-based recombineering, which is facillated by bacteriophage-derived recombinases (lambda-Red and RecET). TargeTron^TM, a group II intron “retrohoming” system, represents the first RNA-guided microbial genome editing tool that has become widely adopted in strains with diminished homologous recombination capabilities such as industrially and biomedically important Clostridium and Staphylococcus. The TargeTron system primarily performs gene knockouts via group II intron insertions, but these intron insertion sites require an elaborate algorithm to find and are distributed in low density. CRISPR-Cas systems are a new generation RNA-guided microbial genome engineering tools that allow targeted genome modifications such as insertions, deletions, and point mutations to be introduced with greater ease than ever before. Here, we demonstrate a bacterial CRISPR/Cas9 toolkit, with and without the lambda-Red recombineering system. This toolkit is shown to facilitate homology-directed repair of double-stranded DNA breaks created by Cas9 endonuclease, creating different types of microbial genetic alterations. Our CRISPR microbial genome engineering system can be used in both gram-negative and gram-positive bacteria, and allows either ssDNA or dsDNA to be used as an editing template. In conclusion, the RNA-guided TargeTron microbial genome engineering system offers researchers a quick and easy method for group II intron insertions leading to gene disruptions; our CRISPR microbial engineering system, with many novel variations, offers researchers even more editing options including deletions, replacements, and point mutations with high efficiency and multi-gene editing capabilities.