title: CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.
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.
authors: Department of Food Science, 1605 Linden Dr, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors: Jee-Hwan
authors: JH
authors: Oh
authors: Department of Food Science, 1605 Linden Dr, University of Wisconsin-Madison, Madison, WI 53706, USA vanpijkeren@wisc.edu.
authors: Jan-Peter
authors: JP
authors: van Pijkeren
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems; D003062:Codon; D004277:DNA, Single-Stranded / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D005818:Genetic Engineering; D016680:Genome, Bacterial; D052200:Limosilactobacillus reuteri / Q000235:genetics*; D016296:Mutagenesis; D009154:Mutation; D011995:Recombination, Genetic
chemical_list: D064130:CRISPR-Associated Proteins; D003062:Codon; D004277:DNA, Single-Stranded; D003851:Deoxyribonucleases