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: 41.51). 01/2013; 31(3). DOI: 10.1038/nbt.2508
Source: PubMed


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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    • "Bacteria Streptococcus pneumoniae And E.coli (Jiang et al., 2013b) "
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    ABSTRACT: CRISPR/Cas, a microbial adaptive immune system, has recently been reshaped as a versatile genome editing approach, endowing genome engineering with high efficiency and robustness. The DNA endonuclease Cas, a component of CRISPR system, is directed to specific target within genomes by guide RNA (gRNA) and performs gene editing function. However, the system is still in its infancy and facing enormous challenges such as off-target mutation. Lots of attempts have been made to overcome such off-targeting and proven to be effective. In this review we focused on recent progress of increasing the CRISPR specificity realized by rational design of gRNA and modification of Cas9 endonuclease. Meanwhile the methods to screen off-target mutation and their effects are also discussed. Comprehensive consideration and rational design to reduce off-target mutation and selection of effective screening assay will greatly facilitate to achieve successful CRISPR/Cas system mediated gene editing.
    Current issues in molecular biology 10/2015; 20:1-12. · 5.75 Impact Factor
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    • "Data generated from the completion of both tasks will be of particular significance to users of strain OV14 and the broader species of E. adhaerens. More specifically, the additional characterization in this study of putative kanamycin-resistant genes provides potential targets for CRISPR/Cas9 genome editing , which has been described as a realistic protocol with which to edit prokaryotic genomes (Jiang et al. 2013). "
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    ABSTRACT: Ensifer adhaerens OV14 underpins the successful crop transformation protocol termed Ensifer-mediated transformation (EMT) but issues exist in regard to addressing the pleomorphic tendency of the bacterium in sub-optimal conditions, identifying the optimal parameters for electro-transformation and defining the strain's antibiotic profile. Here, modifications made to growth medium composition addressed the pleomorphic trait of E. adhaerens OV14; delivering uniform E. adhaerens OV14 growth to ensure efficient rates of electroporation with plasmids up to 42.2 kb in size. Separately, 63 putative antibiotic resistance coding sequences were identified across the E. adhaerens OV14 genome, with testing confirming the strain's susceptibility to gentamicin (≥10 mg L(-1)), tetracycline (≥10 mg L(-1)), chloramphenicol (≥100 mg L(-1)) and cefotaxime (≥500 mg L(-1)) and resistance to ampicillin, paramomycin, streptomycin, spectinomycin, ticarcillin-clavulanate and kanamycin. Partial resistance against carbenicillin, rifampicin, hygromycin-B, and neomycin was also recorded. Resistance to kanamycin was supported by 7 independent nptII-like homologues located within the E. adhaerens OV14 genome. Transcriptional analysis of these targets highlighted two homologues (AHK42288 and AHK42618) whose transcription was significantly elevated within 2 h exposure to kanamycin and which in the case of AHK42288 was maintained out to 6 h post-exposure. In conclusion, our results have identified optimal conditions for the handling of E. adhaerens and have identified a future genome editing target (AHK42288) to negate the kanamycin resistant phenotype of E. adhaerens. © FEMS 2015. All rights reserved. For permissions, please e-mail:
    FEMS Microbiology Letters 08/2015; 362(17). DOI:10.1093/femsle/fnv126 · 2.12 Impact Factor
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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 · 6.77 Impact Factor
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