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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    • "). 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.
    Molecular cell 10/2015; 60(2):242-255. DOI:10.1016/j.molcel.2015.09.020 · 14.02 Impact Factor
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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.
    Journal of Molecular Biology 10/2015; DOI:10.1016/j.jmb.2015.10.004 · 4.33 Impact Factor
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    • "Although there are a few reports on CRISPR-mediated genome editing for bacteria in the current literature, only Type II CRISPR-Cas9 systems have been employed for the purpose. In their pioneer research of conducting bacterial genome editing, Jiang et al. (2013) employed two different plasmids for genome editing in E. coli, one carried the cas9 and the tracrRNA gene of the Streptococcus pyogenes, while the other plasmid carry an artificial CRISPR array (21). The CRISPR-Cas system was introduced into the bacterium in the first transformation while the CRISPR array and donor DNA were co-electroporated into the cas9- carrying E. coli at the second transformation. "
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    ABSTRACT: CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a ther-mophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini-CRISPR array and a donor DNA containing a non-target sequence. Transformation of a pGE plas-mid would yield two alternative fates to transformed cells: wild-type cells are to be targeted for chromoso-mal DNA degradation, leading to cell death, whereas those carrying the mutant gene would survive the cell killing and selectively retained as transformants. Using this strategy, different types of mutation were generated, including deletion, insertion and point mutations. We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference provided the protospacer adjacent motif (PAM) of an un-characterized PAM-dependent CRISPR-Cas system can be predicted by bioinformatic analysis.
    Nucleic Acids Research 10/2015; DOI:10.1093/nar/gkv1044 · 9.11 Impact Factor
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