Work

Streamlined, recombinase-free genome editing with CRISPR-Cas9 in Lactobacillus plantarum reveals barriers to efficient editing

Year: 2018

Type: preprint

Abstract: ABSTRACT Lactic-acid bacteria such as Lactobacillus plantarum are commonly used for fermenting foods and as probiotics, where increasingly sophisticated genome-editing tools are currently being employed to elucidate and enhance these microbes’ beneficial properties. The most advanced tools to-date require heterologous single-stranded DNA recombinases to integrate short oligonucleotides followed by using CRISPR-Cas9 to eliminate cells harboring unedited sequences. Here, we show that encoding the recombineering template on a replicating plasmid allowed efficient genome editing with CRISPR-Cas9 in multiple L. plantarum strains without a recombinase. This strategy accelerated the genome-editing pipeline and could efficiently introduce a stop codon in ribB , silent mutations in ackA , and a complete deletion of lacM . In contrast, oligo-mediated recombineering with CRISPR-Cas9 proved far less efficient in at least one instance. We also observed unexpected outcomes of our recombinase-free method, including an ~1.3-kb genomic deletion when targeting ribB in one strain, and reversion of a point mutation in the recombineering template in another strain. Our method therefore can streamline targeted genome editing in different strains of L. plantarum , although the best means of achieving efficient editing may vary based on the selected sequence modification, gene, and strain.

Source: bioRxiv (Cold Spring Harbor Laboratory)

DOI: 10.1101/352039

Authors: Ryan T. Leenay, Justin M. Vento, Malay Shah, Maria Elena Martino, François Leulier, Chase L. Beisel