CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri. Issue 17 (29th July 2014)

• Record Type: Journal Article
• Authors: Oh, Jee-Hwan; van Pijkeren, Jan-Peter
• Is Part Of: Nucleic acids research. Volume 42:Issue 17(2014)
• Page Start: e131
• Page End: e131
• Publication Date: 2014-07-29
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gku623
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR–Cas systems, such as the Streptococcus pyogenes CRISPR–Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR–Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR–Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR–Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR–Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR–Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR–Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria.