Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems

The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report here the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.

[1]  Zhen Xie,et al.  Improved sgRNA design in bacteria via genome-wide activity profiling , 2018, bioRxiv.

[2]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[3]  Max A. Horlbeck,et al.  Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation , 2014, Cell.

[4]  Qiang Feng,et al.  1,520 reference genomes from cultivated human gut bacteria enable functional microbiome analyses , 2019, Nature Biotechnology.

[5]  Kelly P Williams,et al.  Phylogeny of Gammaproteobacteria , 2010, Journal of bacteriology.

[6]  I. Saito,et al.  Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. , 1998, Gene.

[7]  Brian C. Thomas,et al.  A new view of the tree of life , 2016, Nature Microbiology.

[8]  J. Doudna,et al.  Expanding the Biologist's Toolkit with CRISPR-Cas9. , 2015, Molecular cell.

[9]  A. Goodman,et al.  An insider's perspective: Bacteroides as a window into the microbiome , 2017, Nature Microbiology.

[10]  Adam P. Arkin,et al.  Mutant phenotypes for thousands of bacterial genes of unknown function , 2018, Nature.

[11]  J. Carothers,et al.  Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria , 2018, Nature Communications.

[12]  A. Visel,et al.  CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria , 2019, Nature Microbiology.

[13]  W. Achouak,et al.  Siderophore Typing, a Powerful Tool for the Identification of Fluorescent and Nonfluorescent Pseudomonads , 2002, Applied and Environmental Microbiology.

[14]  M. K. Jensen Design principles for nuclease-deficient CRISPR-based transcriptional regulators , 2018, FEMS Yeast Research.

[15]  M. Lidstrom,et al.  Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. , 2002, BioTechniques.

[16]  Dongchang Sun Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria , 2018, Front. Microbiol..

[17]  Nikhil U. Nair,et al.  Toward a genetic tool development pipeline for host-associated bacteria. , 2017, Current opinion in microbiology.

[18]  Finbarr Hayes,et al.  Transposon-based strategies for microbial functional genomics and proteomics. , 2003, Annual review of genetics.

[19]  Jan P. Meier-Kolthoff,et al.  Correction for Barka et al., Taxonomy, Physiology, and Natural Products of Actinobacteria , 2016, Microbiology and Molecular Reviews.

[20]  Huimin Zhao,et al.  CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973 , 2016, Microbial Cell Factories.

[21]  Nikhil U. Nair,et al.  Synthetic Biology Approaches to Engineer Probiotics and Members of the Human Microbiota for Biomedical Applications. , 2018, Annual review of biomedical engineering.

[22]  Heng Li,et al.  Minimap2: pairwise alignment for nucleotide sequences , 2017, Bioinform..

[23]  A. Deutschbauer,et al.  Rapidly moving new bacteria to model-organism status. , 2018, Current opinion in biotechnology.

[24]  Yang Gu,et al.  CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii. , 2016, Biotechnology journal.

[25]  Mazhar Adli,et al.  The CRISPR tool kit for genome editing and beyond , 2018, Nature Communications.

[26]  Feng Zhang,et al.  CRISPR-assisted editing of bacterial genomes , 2013, Nature Biotechnology.

[27]  S. Smolinski,et al.  Building a genome engineering toolbox in nonmodel prokaryotic microbes , 2018, Biotechnology and bioengineering.

[28]  J. Altenbuchner Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System , 2016, Applied and Environmental Microbiology.

[29]  K. Thormann,et al.  Two different stator systems drive a single polar flagellum in Shewanella oneidensis MR‐1 , 2009, Molecular microbiology.

[30]  N. I. López,et al.  Glycerol inhibition of melanin biosynthesis in the environmental Aeromonas salmonicida 34melT , 2018, Applied Microbiology and Biotechnology.

[31]  V. Martin,et al.  The isolation of DNA sequences flanking Tn5 transposon insertions by inverse PCR. , 2002, Methods in molecular biology.

[32]  Xin Yan,et al.  Cre/lox System and PCR-Based Genome Engineering in Bacillus subtilis , 2008, Applied and Environmental Microbiology.

[33]  K. Yamamura,et al.  Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites. , 2002, Nucleic acids research.

[34]  Chase L. Beisel,et al.  Barriers to genome editing with CRISPR in bacteria , 2019, Journal of Industrial Microbiology & Biotechnology.

[35]  Byung-Kwan Cho,et al.  Applications of CRISPR/Cas System to Bacterial Metabolic Engineering , 2018, International journal of molecular sciences.

[36]  Manish Kushwaha,et al.  A portable expression resource for engineering cross-species genetic circuits and pathways , 2015, Nature Communications.

[37]  Luke A. Gilbert,et al.  Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression , 2013, Cell.

[38]  J. Tommassen,et al.  Unearthing the genomes of plant-beneficial Pseudomonas model strains WCS358, WCS374 and WCS417 , 2015, BMC Genomics.

[39]  Adrian Pickar-Oliver,et al.  The next generation of CRISPR–Cas technologies and applications , 2019, Nature Reviews Molecular Cell Biology.

[40]  I-Min A. Chen,et al.  IMG-ABC: new features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes , 2016, Nucleic Acids Res..