Development and characterization of a CRISPR/Cas9n-based multiplex genome editing system for Bacillus subtilis

Metabolic engineering has expanded from a focus on designs requiring a small number of genetic modifications to increasingly complex designs driven by advances in multiplex genome editing technologies. However, simultaneously modulating multiple genes on the chromosome remains challenging in Bacillus subtilis. Thus, developing an efficient and convenient method for B. subtilis multiplex genome editing is imperative. Here, we developed a CRISPR/Cas9n-based multiplex genome editing system for iterative genome editing in B. subtilis. This system enabled us to introduce various types of genomic modifications with more satisfying efficiency than using CRISPR/Cas9, especially in multiplex gene editing. Our system achieved at least 80% efficiency for 1–8 kb gene deletions, at least 90% efficiency for 1–2 kb gene insertions, near 100% efficiency for site-directed mutagenesis, 23.6% efficiency for large DNA fragment deletion and near 50% efficiency for three simultaneous point mutations. The efficiency for multiplex gene editing was further improved by regulating the nick repair mechanism mediated by ligD gene, which finally led to roughly 65% efficiency for introducing three point mutations on the chromosome. To demonstrate its potential, we applied our system to simultaneously fine-tune three genes in the riboflavin operon and significantly improved the production of riboflavin in a single cycle. We present not only the iterative CRISPR/Cas9n system for B. subtilis but also the highest efficiency for simultaneous modulation of multiple genes on the chromosome in B. subtilis reported to date. We anticipate this CRISPR/Cas9n mediated system to greatly enhance the optimization of diverse biological systems via metabolic engineering and synthetic biology.

[1]  M. Moo-young,et al.  Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis , 2016, Applied and Environmental Microbiology.

[2]  K. Kuroda,et al.  Precise genome-wide base editing by the CRISPR Nickase system in yeast , 2017, Scientific Reports.

[3]  B. Joris,et al.  New Integrative Method To Generate Bacillus subtilis Recombinant Strains Free of Selection Markers , 2004, Applied and Environmental Microbiology.

[4]  J. Keasling,et al.  CRISPR/Cas9 advances engineering of microbial cell factories. , 2016, Metabolic engineering.

[5]  C. Barbas,et al.  ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. , 2013, Trends in biotechnology.

[6]  Sang Yup Lee,et al.  CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum. , 2017, Metabolic engineering.

[7]  J. Altenbuchner,et al.  New CRISPR‐Cas9 vectors for genetic modifications of Bacillus species , 2018, FEMS microbiology letters.

[8]  N. Ogasawara,et al.  A simple method for introducing marker-free deletions in the Bacillus subtilis genome. , 2011, Methods in molecular biology.

[9]  Christopher A. Voigt,et al.  Automated Design of Synthetic Ribosome Binding Sites to Precisely Control Protein Expression , 2009, Nature Biotechnology.

[10]  C. Anagnostopoulos,et al.  REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS , 1961, Journal of bacteriology.

[11]  G. Reitz,et al.  Role of DNA Repair by Nonhomologous-End Joining in Bacillus subtilis Spore Resistance to Extreme Dryness, Mono- and Polychromatic UV, and Ionizing Radiation , 2007, Journal of bacteriology.

[12]  Tao Chen,et al.  Construction, Model-Based Analysis, and Characterization of a Promoter Library for Fine-Tuned Gene Expression in Bacillus subtilis. , 2018, ACS synthetic biology.

[13]  Tao Chen,et al.  Integrated whole-genome and transcriptome sequence analysis reveals the genetic characteristics of a riboflavin-overproducing Bacillus subtilis. , 2018, Metabolic engineering.

[14]  B. Stoddard,et al.  Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template , 2010, Nucleic acids research.

[15]  Ryan T Gill,et al.  Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides , 2010, Nature Biotechnology.

[16]  J. Keasling,et al.  Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development , 2014, Nature Reviews Microbiology.

[17]  Y. Yoshikuni,et al.  Implementation of stable and complex biological systems through recombinase-assisted genome engineering , 2013, Nature Communications.

[18]  Z. Cui,et al.  mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis , 2006, Nucleic acids research.

[19]  John S. Hawkins,et al.  Bacterial CRISPR: accomplishments and prospects. , 2015, Current opinion in microbiology.

[20]  Kunqiang Hong,et al.  Recent advances in CRISPR/Cas9 mediated genome editing in Bacillus subtilis , 2018, World Journal of Microbiology and Biotechnology.

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

[22]  P. K. Ajikumar,et al.  The future of metabolic engineering and synthetic biology: towards a systematic practice. , 2012, Metabolic engineering.

[23]  S. Park,et al.  A Highly Efficient CRISPR-Cas9-Mediated Large Genomic Deletion in Bacillus subtilis , 2017, Front. Microbiol..

[24]  Guocheng Du,et al.  Metabolic engineering of Bacillus subtilis fueled by systems biology: Recent advances and future directions. , 2017, Biotechnology advances.

[25]  Xueming Zhao,et al.  Deregulation of purine pathway in Bacillus subtilis and its use in riboflavin biosynthesis , 2014, Microbial Cell Factories.

[26]  G. Reitz,et al.  Impact of two DNA repair pathways, homologous recombination and non-homologous end joining, on bacterial spore inactivation under simulated martian environmental conditions , 2011 .

[27]  Sheng Yang,et al.  CRISPR-Cas9D10A Nickase-Assisted Genome Editing in Lactobacillus casei , 2017, Applied and Environmental Microbiology.

[28]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[29]  N. Maizels,et al.  DNA Nicks Promote Efficient and Safe Targeted Gene Correction , 2011, PloS one.

[30]  Hal S. Alper,et al.  A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae , 2014, BMC Systems Biology.

[31]  Shihui Yang,et al.  CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in Bacillus subtilis , 2019, Nucleic acids research.

[32]  Merja Penttilä,et al.  Yeast oligo-mediated genome engineering (YOGE). , 2013, ACS synthetic biology.

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

[34]  Jing Liang,et al.  Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision , 2018, Nature Biotechnology.

[35]  E. Lander,et al.  Genetic Screens in Human Cells Using the CRISPR-Cas9 System , 2013, Science.

[36]  Zhili He,et al.  Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase , 2018, Applied and Environmental Microbiology.

[37]  Huimin Zhao,et al.  Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. , 2015, ACS synthetic biology.

[38]  Brian F. Pfleger,et al.  Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes , 2006, Nature Biotechnology.

[39]  P. Jeggo,et al.  Identification of a DNA Nonhomologous End-Joining Complex in Bacteria , 2002, Science.

[40]  Xueli Zhang,et al.  The CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway. , 2017, Metabolic engineering.

[41]  Yan Zhang,et al.  Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. , 2015, Metabolic engineering.

[42]  Farren J. Isaacs,et al.  Programming cells by multiplex genome engineering and accelerated evolution , 2009, Nature.

[43]  Jingdong Tian,et al.  Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries , 2011, Nature Protocols.

[44]  Hilde van der Togt,et al.  Publisher's Note , 2003, J. Netw. Comput. Appl..

[45]  Ryan T Gill,et al.  Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering , 2016, Nature Biotechnology.

[46]  S. Park,et al.  Genome engineering using a synthetic gene circuit in Bacillus subtilis , 2014, Nucleic acids research.

[48]  Meagan E. Sullender,et al.  Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 , 2015, Nature Biotechnology.

[49]  S. Lee,et al.  CRISPR technologies for bacterial systems: Current achievements and future directions. , 2016, Biotechnology advances.

[50]  C. Tomlin,et al.  Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay , 2013, Nucleic acids research.

[51]  Huimin Zhao,et al.  FairyTALE: a high-throughput TAL effector synthesis platform. , 2014, ACS synthetic biology.

[52]  Sheng Yang,et al.  Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System , 2015, Applied and Environmental Microbiology.

[53]  X. Duan,et al.  Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system , 2016, Scientific Reports.

[54]  N. Ogasawara,et al.  A new simple method to introduce marker-free deletions in the Bacillus subtilis genome. , 2009, Genes & genetic systems.

[55]  Christopher L. Hemme,et al.  Efficient Genome Editing in Clostridium cellulolyticum via CRISPR-Cas9 Nickase , 2015, Applied and Environmental Microbiology.

[56]  J C Rabinowitz,et al.  The influence of ribosome‐binding‐site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo , 1992, Molecular microbiology.

[57]  Guido Grandi,et al.  Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli , 2017, Microbial Cell Factories.

[58]  T. Helleday,et al.  DNA double-strand break repair: from mechanistic understanding to cancer treatment. , 2007, DNA repair.

[59]  Q. Shen,et al.  Bacillus subtilis genome editing using ssDNA with short homology regions , 2012, Nucleic acids research.

[60]  Zhiwen Wang,et al.  Establishment of a Markerless Mutation Delivery System in Bacillus subtilis Stimulated by a Double-Strand Break in the Chromosome , 2013, PloS one.

[61]  Lei S. Qi,et al.  CRISPR/Cas9 in Genome Editing and Beyond. , 2016, Annual review of biochemistry.

[62]  Christoph Wittmann,et al.  Advanced biotechnology: metabolically engineered cells for the bio-based production of chemicals and fuels, materials, and health-care products. , 2015, Angewandte Chemie.