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The CRISPR-Cas system of Streptococcus pyogenes: function and applications

Andrew Varble, Luciano Marraffini

Here we review how the Cas9 nuclease mediates anti-phage immunity and how it can be repurposed for the genetic engineering of human cells and other eukaryotic organisms.

Cleavage of viral DNA by restriction endonucleases stimulates the type II CRISPR-Cas immune response

Pascal Maguin, Andrew Varble, Joshua W Modell, Luciano A Marraffini

We found that restriction endonucleases provide a short-term defense, which is rapidly overcome through methylation of the phage genome. In a small fraction of the cells, however, restriction results in the acquisition of spacer sequences from the cleavage site, which mediates a robust type II-A CRISPR-Cas immune response against the methylated phage


Prophage integration into CRISPR loci enables evasion of antiviral immunity in Streptococcus pyogenes

Andrew Varble, Edmondo Campisi, Chad W Euler, Pascal Maguin, Albina Kozlova, Jessica Fyodorova, Jakob T Rostøl, Vincent A Fischetti, Luciano A Marraffini

Here we demonstrate that the ΦAP1.1 temperate phage utilizes an alternative approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection, this phage expresses a small anti-CRISPR protein, AcrIIA23, that prevents Cas9 function, allowing ΦAP1.1 to integrate into the direct repeats of the CRISPR locus, neutralizing immunity.

RecT Recombinase Expression Enables Efficient Gene Editing in Enterococcus spp

Victor Chen, Matthew E Griffin, Pascal Maguin, Andrew Varble, Howard C Hang

The dissection of E. faecium functions and mechanisms has been restricted by inefficient gene-editing methods. To address these limitations, here, we report that the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae.

Type III-A CRISPR immunity promotes mutagenesis of staphylococci

Charlie Y Mo, Jacob Mathai, Jakob T Rostøl, Andrew Varble,, Dalton V Banh, Luciano A Marraffini

Here we show that the non-specific DNase activity of the staphylococcal type III-A CRISPR-Cas system increases mutations in the host and accelerates the generation of antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis.


Three New Cs for CRISPR: Collateral, Communicate, Cooperate

Andrew Varble, Luciano A Marraffini

Recent work in the field has revealed unexpected features of the CRISPR-Cas mechanism: (i) collateral, nonspecific, cleavage of host nucleic acids; (ii) secondary messengers that amplify the immune response; and (iii) immunosuppression of CRISPR targeting by phage-encoded inhibitors. Here, we review these new and exciting findings.

Recombination between phages and CRISPR-cas loci facilitates horizontal gene transfer in staphylococci

Andrew Varble, Sean Meaden, Rodolphe Barrangou, Edze R Westra, Luciano A Marraffini

Although CRISPR-cas loci are widely distributed throughout microbial genomes and often display hallmarks of horizontal gene transfer10-12, the drivers of CRISPR dissemination remain unclear. Here, we show that spacers can recombine with phage target sequences to mediate a form of specialized transduction of CRISPR elements


Incomplete prophage tolerance by type III-A CRISPR-Cas systems reduces the fitness of lysogenic hosts

Gregory W Goldberg, Elizabeth A McMillan, Andrew Varble, Joshua W Modell, Poulami Samai, Wenyan Jiang, Luciano A Marraffini

Here we show that maintenance of conditionally tolerant type III-A systems can produce fitness costs within populations of Staphylococcus aureus lysogens. 

Broad Targeting Specificity during Bacterial Type III CRISPR-Cas Immunity Constrains Viral Escape

Nora C Pyenson, Kaitlyn Gayvert, Andrew Varble, Olivier Elemento, Luciano A Marraffini

Here we show that targeting by the Staphylococcus epidermidis type III-A CRISPR-Cas system does not require PAM or seed sequences, and thus prevents viral escape via single-nucleotide substitutions. Instead, viral escapers can only arise through complete target deletion.