RNA-guided editing of bacterial genomes using CRISPR-Cas systems

1] Laboratory of Bacteriology, The Rockefeller University, New York, New York, USA. [2].
Nature Biotechnology (Impact Factor: 39.08). 01/2013; 31(3). DOI: 10.1038/nbt.2508
Source: PubMed

ABSTRACT Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.

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    • "In E. coli, Jiang et al. (2013) was the first to report CRISPR–Cas9 mediated genome editing. The authors used ssDNA as editing template and achieved 65% efficiency for introducing a codon replacement. "
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    ABSTRACT: Engineering cellular metabolism for improved production of valuable chemicals requires extensive modulation of bacterial genome to explore complex genetic spaces. Here, we report the development of a CRISPR-Cas9 based method for iterative genome editing and metabolic engineering of Escherichia coli. This system enables us to introduce various types of genomic modifications with near 100% editing efficiency and to introduce three mutations simultaneously. We also found that cells with intact mismatch repair system had reduced chance to escape CRISPR mediated cleavage and yielded increased editing efficiency. To demonstrate its potential, we used our method to integrate the β-carotene synthetic pathway into the genome and to optimize the methylerythritol-phosphate (MEP) pathway and central metabolic pathways for β-carotene overproduction. We collectively tested 33 genomic modifications and constructed more than 100genetic variants for combinatorially exploring the metabolic landscape. Our best producer contained15 targeted mutations and produced 2.0g/L β-carotene in fed-batch fermentation. Copyright © 2015. Published by Elsevier Inc.
    Metabolic Engineering 06/2015; 31. DOI:10.1016/j.ymben.2015.06.006 · 8.26 Impact Factor
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    • "In type-II CRISPR/Cas systems, Cas9 forms a dual-RNA complex with crRNA and a transactivating CRISPR-RNA (tracrRNA) that is required for Cas9 nuclease to be functional (Chylinski et al., 2013). Recently, the natural CRISPR/Cas9 system from Streptococcus pyogenes containing the dual-RNA (crRNA and tracrRNA) and Cas9 has been applied for genome engineering in both Gram-positive S. pneumonia (Jiang et al., 2013) and Lactobacillus reuteri (Oh and van Pijkeren, 2014) and Gram-negative E. coli (Jiang et al., 2013) bacteria. In all cases, the host strain needs to have an established functional recombineering system if it is not highly recombinogenic; the CRISPR/Cas9 system is primarily functional as a tool for selecting edited cells against non-edited background cells (with a possible modest induction of recombination) thereby leading to high efficiency of genome engineering. "
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    ABSTRACT: The anaerobic spore-forming, gram-positive, solventogenic clostridia are notorious for being difficult to genetically engineer. Based on CRISPR/Cas9 assisted homologous recombination, we demonstrated that clean markerless gene deletion from the chromosome can be easily achieved with a high efficiency through a single-step transformation in Clostridium beijerinckii NCIMB 8052, one of the most prominent strains for acetone, butanol and ethanol (ABE) production. This highly efficient genome engineering system can be further explored for multiplex genome engineering purposes. The protocols and principles developed in this study provided valuable references for genome engineering in other microorganisms lacking developed genetic engineering tools. Copyright © 2015. Published by Elsevier B.V.
    Journal of Biotechnology 02/2015; 10. DOI:10.1016/j.jbiotec.2015.02.005 · 2.88 Impact Factor
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    • "Consequently, the spacer in the mature crRNA matches only 20 of the 30-nt protospacer sequence in the invading nucleic acid. The non-matching fragment in the protospacer is not important for the CRISPRmediated immunity; however, shortening of the protospacer sequence to 19nt or more abrogates CRISPR-mediated plasmid interference (Gasiunas et al., 2012; Jiang et al., 2013b). Three model systems have been used to study mechanisms of invading nucleic acid destruction by Type II systems (Gasiunas et al., 2014). "
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    ABSTRACT: Approximately all sequenced archaeal and half of eubacterial genomes have some sort of adaptive immune system, which enables them to target and cleave invading foreign genetic elements by an RNAi-like pathway. CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems consist of the CRISPR loci with multiple copies of a short repeat sequence separated by variable sequences with similar size that are derived from invaders and cas genes encode proteins involved in RNA binding, endo-and exo-nucleases, helicases, and polymerases activities. There are three main types (I, II and III) of CRISPR/Cas systems. All systems function in three distinct stages: (1) adaptation, (2) crRNA biogenesis, and (3) interference. This review focuses on the features and mechanisms of the CRISPR-Cas systems and current finding about them.
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