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Nucleic Acids Res. 2014;42(17):e131. doi: 10.1093/nar/gku623. Epub 2014 Jul 29.

CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.

Author information

1
Department of Food Science, 1605 Linden Dr, University of Wisconsin-Madison, Madison, WI 53706, USA.
2
Department of Food Science, 1605 Linden Dr, University of Wisconsin-Madison, Madison, WI 53706, USA vanpijkeren@wisc.edu.

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.

PMID:
25074379
PMCID:
PMC4176153
DOI:
10.1093/nar/gku623
[Indexed for MEDLINE]
Free PMC Article

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