Background

CRISPR-Cas (Clustered, regularly interspaced, short palindromic repeats-CRISPR associated) is a prokaryotic adaptive immune system found in almost 90% of sequenced archaea and about 40% of bacteria (Makarova et al., 2015). These systems recognize and destroy nucleic acid invaders in a sequence-specific manner (van der Oost et al., 2014). A CRISPR locus typically contains an array of short DNA repeats (~35 nucleotides in length) separated by equally-short unique sequences called spacers, which are often derived from mobile genetic elements. The repeats and spacers are transcribed and processed into small CRISPR RNAs (crRNAs) that each specifies a single target. crRNAs assemble with one or more Cas proteins to form an effector complex that recognizes and degrades nucleic acids that bear a sequence, called a protospacer, complementary to the crRNA. Depending on the CRISPR-Cas type present in an organism, other limitations exist on target recognition, such as the presence of a short protospacer-adjacent motif (PAM) required for targeting by Type I and II systems (Mojica et al., 2009) or the requirement in Type III systems for specific base-pair mismatches between the crRNA and the target (Marraffini and Sontheimer, 2010). Nucleic acid invaders targeted by CRISPR-Cas are typically bacteriophages or mobile genetic elements such as plasmids or transposons that often carry virulence factors, including antibiotic resistance genes and toxins (Novick, 2003; Liu et al., 2016). CRISPR-Cas is thus a potential major barrier to the spread of virulence factors within prokaryotes and has been shown to prevent conjugative transfer of antibiotic resistance plasmids within human clinical isolates of Staphylococcus species (Marraffini and Sontheimer, 2008). Some staphylococci, including human pathogenic isolates of S. epidermidis and S. aureus, the two Staphylococcus species most commonly found in human infections, carry CRISPR-Cas systems (Cao et al., 2016; Li et al., 2016). Within these strains, multiple CRISPR spacers have been found that naturally bear homology to mobile staphylococcal plasmids, indicating that these organisms are capable of using CRISPR-Cas to limit the spread of virulence factors (Samai et al., 2015; Li et al., 2016). Investigations of the effect of mutations within the cas genes and the repeat-spacer array in Staphylococcus on anti-plasmid immunity have provided key insights into the function and mechanisms of Type III-A CRISPR-Cas in S. epidermidis RP62a (Hatoum-Aslan et al., 2011 and 2014; Maniv et al., 2016). Notably, similar assays have been used for mechanistic CRISPR-Cas studies in other organisms, including Enterococcus faecalis, Escherichia coli, and Listeria monocytogenes (Richter et al., 2014; Sesto et al., 2014; Price et al., 2016). Other methods of quantifying CRISPR anti-plasmid immunity, namely transformation via electroporation, have been used (Cao et al., 2016); however, this method cannot be used in non-competent or weakly/selectively competent organisms such as L. monocytogenes and many strains of Staphylococcus (Monk et al., 2012; Sesto et al., 2014). In these organisms, conjugation assays provide not only a viable alternative, but also a more physiologically relevant means of testing CRISPR-Cas anti-plasmid immunity. Described below is a quantitative method to determine the efficacy of CRISPR-mediated interference of the transfer of conjugative plasmid pG0400 between S. aureus and S. epidermidis. While this protocol focuses on staphylococci, it could be adapted for any prokaryotes capable of conjugation.