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Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition.

ABSTRACT: While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L. gasseri and found that it has modest activity in a cell-free lysate assay but no activity in mammalian cells even when altering promoter, position of tag sequences and NLS, and length of crRNA:tracrRNA. In the lysate assay we tested over 400 sequential crRNA target sequences and found that the Lga Cas9 PAM is NNGA/NDRA, different than NTAA predicted from the native bacterial host. In addition, we found multiple instances of consecutive crRNA target sites, indicating flexibility in either PAM sequence or distance from the crRNA target site. This work highlights the need for characterization of new CRISPR systems and highlights the non-triviality of porting them into eukaryotes as gene editing tools.

SUBMITTER: Anderson EM

PROVIDER: S-EPMC5796720 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition.

Anderson Emily M EM McClelland Shawn S Maksimova Elena E Strezoska Žaklina Ž Basila Megan M Briner Alexandra E AE Barrangou Rodolphe R Smith Anja van Brabant AVB

PloS one 20180202 2

While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L. gasseri and found that it has modest activity in a cell-free lysate assay but no activity in mammalian cells even when altering promoter, position of tag sequences and NLS, and length of crRNA:tracrRNA. In the lysate assay we tested over 400 sequential crRNA targ ...[more]

PMID: 29394276

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Project description:Diverse Lactobacillus strains are widely used as probiotic cultures in the dairy and dietary supplement industries, and specific strains, such as Lactobacillus acidophilus NCFM, have been engineered for the development of biotherapeutics. To expand the Lactobacillus manipulation toolbox with enhanced efficiency and ease, we present here a CRISPR (clustered regularly interspaced palindromic repeats)-SpyCas9D10A nickase (Cas9N)-based system for programmable engineering of L. acidophilus NCFM, a model probiotic bacterium. Successful single-plasmid delivery system was achieved with the engineered pLbCas9N vector harboring cas9N under the regulation of a Lactobacillus promoter and a cloning region for a customized single guide RNA (sgRNA) and editing template. The functionality of the pLbCas9N system was validated in NCFM with targeted chromosomal deletions ranging between 300 bp and 1.9 kb at various loci (rafE, lacS, and ltaS), yielding 35 to 100% mutant recovery rates. Genome analysis of the mutants confirmed precision and specificity of the pLbCas9N system. To showcase the versatility of this system, we also inserted an mCherry fluorescent-protein gene downstream of the pgm gene to create a polycistronic transcript. The pLbCas9N system was further deployed in other species to generate a concurrent single-base substitution and gene deletion in Lactobacillus gasseri ATCC 33323 and an in-frame gene deletion in Lactobacillus paracasei Lpc-37, highlighting the portability of the system in phylogenetically distant Lactobacillus species, where its targeting activity was not interfered with by endogenous CRISPR-Cas systems. Collectively, these editing outcomes illustrate the robustness and versatility of the pLbCas9N system for genome manipulations in diverse lactobacilli and open new avenues for the engineering of health-promoting lactic acid bacteria.IMPORTANCE This work describes the development of a lactobacillus CRISPR-based editing system for genome manipulations in three Lactobacillus species belonging to the lactic acid bacteria (LAB), which are commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiotas and for their emerging roles in human and animal commercial health-promoting applications. We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in Lactobacillus acidophilus and further demonstrated the efficacy of this universal system in two distantly related Lactobacillus species. This versatile Cas9-based system facilitates genome engineering compared to conventional gene replacement systems and represents a valuable gene editing modality in species that do not possess native CRISPR-Cas systems. Overall, this portable tool contributes to expanding the genome editing toolbox of LAB for studying their health-promoting mechanisms and engineering of these beneficial microbes as next-generation vaccines and designer probiotics.

Project description:CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioides (Clostridium) difficile, a major human enteropathogen, and showed that it could be harnessed for efficient genome editing in this bacterium. However, molecular details are still missing on CRISPR-Cas function for adaptation and sequence requirements for both interference and new spacer acquisition in this pathogen. Despite accumulating knowledge on the individual CRISPR-Cas systems in various prokaryotes, no data are available on the adaptation process in bacterial type I-B CRISPR-Cas systems. Here, we report the first experimental evidence that the C. difficile type I-B CRISPR-Cas system acquires new spacers upon overexpression of its adaptation module. The majority of new spacers are derived from a plasmid expressing Cas proteins required for adaptation or from regions of the C. difficile genome where generation of free DNA termini is expected. Results from protospacer-adjacent motif (PAM) library experiments and plasmid conjugation efficiency assays indicate that C. difficile CRISPR-Cas requires the YCN consensus PAM for efficient interference. We revealed a functional link between the adaptation and interference machineries, since newly adapted spacers are derived from sequences associated with a CCN PAM, which fits the interference consensus. The definition of functional PAMs and establishment of relative activity levels of each of the multiple C. difficile CRISPR arrays in present study are necessary for further CRISPR-based biotechnological and medical applications involving this organism. IMPORTANCE CRISPR-Cas systems provide prokaryotes with adaptive immunity for defense against foreign nucleic acid invaders, such as viruses or phages and plasmids. The CRISPR-Cas systems are highly diverse, and detailed studies of individual CRISPR-Cas subtypes are important for our understanding of various aspects of microbial adaptation strategies and for the potential applications. The significance of our work is in providing the first experimental evidence for type I-B CRISPR-Cas system adaptation in the emerging human enteropathogen Clostridioides difficile. This bacterium needs to survive in phage-rich gut communities, and its active CRISPR-Cas system might provide efficient antiphage defense by acquiring new spacers that constitute memory for further invader elimination. Our study also reveals a functional link between the adaptation and interference CRISPR machineries. The definition of all possible functional trinucleotide motifs upstream protospacers within foreign nucleic acid sequences is important for CRISPR-based genome editing in this pathogen and for developing new drugs against C. difficile infections.

Dataset Information

Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition.

Publications

Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition.

Similar Datasets

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