Identification and functional study of type III-A CRISPR-Cas systems in clinical isolates of Staphylococcus aureus

RNA activation‐independent DNA targeting of the Type III CRISPR‐Cas system by a Csm complex

“…In contrast, the Type I and Type II systems target exclusively DNA, and the Type III system targets RNA or/and DNA [3,[17][18][19][20][21][22] The Type III system can be further classified into subtypes (subtypes III-A-III-D) or mainly into Type III-A and Type III-B [14,23]. The Csm complexes of Type III-A from Sulfolobus solfataricus, Thermus thermophilus, Streptococcus thermophilus, Staphylococcus aureus and Staphylococcus epidermis have been characterized in vitro and in vivo [3,19,[21][22][23][24]. The Csm complex consists of five subunits (Csm1-5) and a crRNA, while the Cmr complex of Type III-B consists of six subunits (Cmr1-6) and a crRNA [22,25,26].…”

Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci

“…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).…”

“…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.…”

Expression and Purification of the Cas10-Csm Complex from Staphylococci

“…These repeat-spacer arrays encode small CRISPR RNAs (crRNAs) that associate with Cas proteins, forming a ribonucleoprotein complex that destroys foreign DNA and/or RNA in a sequence-dependent manner. Many staphylococci possess Type III-A CRISPR-Cas systems (Marraffini and Sontheimer, 2008; Golding et al ., 2012; Cao et al ., 2016). The Type III-A system in S. epidermidis RP62a, a wild-type human isolate (Christensen et al ., 1987), encodes a multi-subunit complex called Cas10-Csm, composed of Cas10, Csm2, Csm3, Csm4, Csm5 and a crRNA (Hatoum-Aslan et al ., 2013).…”

“…Staphylococcus epidermidis and Staphylococcus aureus are bacterial residents on human skin that are also leading causes of antibiotic resistant infections (Lowy, 1998; National Nosocomial Infections Surveillance, 2004; Otto, 2009). Many staphylococci possess Type III-A CRISPR-Cas systems (Marraffini and Sontheimer, 2008; Cao et al ., 2016), which have been shown to prevent plasmid transfer and protect against viral predators (Goldberg et al ., 2014; Hatoum-Aslan et al ., 2014; Samai et al ., 2015) in these organisms. Thus, gaining a mechanistic understanding of these systems in the native staphylococcal background can lead to important insights into the factors that impact the evolution and survival of these pathogens.…”