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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    • "To find a suitable target site, ZFNs require software-assistance, TALENs have no such limitations (Voytas 2013), whereas CRISPR-Cas requires a simple target sequence, which is GN 19 NGG when using the archetypal S. pyogenes-based technology with an U6 promoter-driven sgRNA (Belhaj et al. 2013). This CRISPR- Cas target site includes a 5′ guide RNA-binding region and a 3′ protospacer-adjacent motif (PAM), which is an NGG (Cong et al. 2013; Mali et al. 2013b) or NAG sequence (Mali et al. 2013a; Jiang et al. 2013a), and affords abundant targets (protospacers) for cleavage. Targeted mutagenesis by nucleases is a valuable tool in reverse genetics, and has some advantages, in terms of causing permanent and complete loss-of-function, over RNA interference (Puchta and Fauser 2014). "
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    ABSTRACT: Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining β-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a β-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (−1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k cat, but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k cat). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with ~10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.
    Full-text · Article · Dec 2015 · Applied Microbiology and Biotechnology
    • "). Internalized ssDNAs are coated with RecA and ssDNA-binding proteins that facilitate homology search, and we show that these proteins do not inhibit NmeCas9 activity in vitro. It is not known which of these stages of transformation are subject to CRISPR interference , though genomic dsDNA is clearly susceptible (Bikard et al., 2012; Jiang et al., 2013; Vercoe et al., 2013). In addition, either DNA strand can be randomly internalized during transformation , yet a crRNA that is complementary to only one strand completely blocks transformation (Bikard et al., 2012; Zhang et al., 2013). "
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    ABSTRACT: Type II CRISPR systems defend against invasive DNA by using Cas9 as an RNA-guided nuclease that creates double-stranded DNA breaks. Dual RNAs (CRISPR RNA [crRNA] and tracrRNA) are required for Cas9's targeting activities observed to date. Targeting requires a protospacer adjacent motif (PAM) and crRNA-DNA complementarity. Cas9 orthologs (including Neisseria meningitidis Cas9 [NmeCas9]) have also been adopted for genome engineering. Here we examine the DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns. Unexpectedly, NmeCas9 cleaves single-stranded DNAs in a manner that is RNA guided but PAM and tracrRNA independent. Beyond the need for guide-target pairing, this "DNase H" activity has no apparent sequence requirements, and the cleavage sites are measured from the 5' end of the DNA substrate's RNA-paired region. These results indicate that tracrRNA is not strictly required for NmeCas9 enzymatic activation, and expand the list of targeting activities of Cas9 endonucleases.
    No preview · Article · Oct 2015 · Molecular cell
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    • "It is this function that makes adapted CRISPR systems such attractive tools for genetic engineering. The Cas9 protein from the Streptococcus pyogenes type II CRISPR system has been widely applied as a minimal functional unit for the recognition and cleavage of target double-stranded DNA for genome engineering [90] [91] [92] [93] [94] and in its mutant endonuclease-inactivated form dCas9 as a DNA binding protein [19] [33]. Cas9 requires both a guide RNA and a tracrRNA (trans-activating CRISPR RNA) to function; the guide RNA can be expressed either as a crRNA (CRISPR RNA) as it is from the CRISPR array or as a fusion to the tracrRNA known as an sgRNA (small guide RNA) [95] (Fig. 1c-i). "
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    ABSTRACT: Synthetic biologists aim to construct novel genetic circuits with useful applications through rational design and forward engineering. Given the complexity of signal processing that occurs in natural biological systems, engineered microbes have the potential to perform a wide range of desirable tasks which require sophisticated computation and control. Realising this goal will require accurate predictive design of complex synthetic gene circuits and accompanying large sets of quality modular and orthogonal genetic parts. Here we present a current overview of the versatile components and tools available for engineering gene circuits in microbes, including recently developed RNA-based tools that possess large dynamic ranges and can be easily programmed. We introduce design principles that enable robust and scalable circuit performance such as insulating a gene circuit against unwanted interactions with its context, and describe efficient strategies for rapidly identifying and correcting causes of failure and fine tuning circuit characteristics.
    Full-text · Article · Oct 2015 · Journal of Molecular Biology
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