CRISPR-Cas Systems in Prokaryotes

Abstract

A b s t r a c tProkaryotic organisms possess numerous strategies that enable survival in hostile conditions. Among others, these conditions include the invasion of foreign nucleic acids such as bacteriophages and plasmids. The clustered regularly interspaced palindromic repeats-CRISPRassociated proteins (CRISPR-Cas) system provides the majority of bacteria and archaea with adaptive and hereditary immunity against this threat. This mechanism of immunity is based on short fragments of foreign DNA incorporated within the hosts genome. After transcription, these fragments guide protein complexes that target foreign nucleic acids and promote their degradation. The aim of this review is to summarize the current status of CRISPR-Cas research, including the mechanisms of action, the classification of different types and subtypes of these systems, and the development of new CRISPR-Cas-based molecular biology tools. K e y w o r d s: CRISPR-Cas, prokaryotesBurmistrz M. and Pyrć K. 3 194 spacer sequences can be of either foreign or self-origin (Stern et al., 2010;Vercoe et al., 2013). The proteomic component is responsible for the incorporation of new template sequences, processing them into a form that enables base pairing with target nucleic acids, as well as for scanning and cleavage of target DNA or RNA. Of note, not all CRISPR-Cas systems discovered to date are active (Haft et al., 2005;van der Ploeg, 2009). Structure of the CRISPR-Cas systemThe genomic component of the CRISPR-Cas system is formed by a series of variable spacers, which in some cases share sequence similarity with viruses, plasmids, or bacteria. These regions are interspaced with repeat sequences that are identical or almost identical within a single CRISPR cassette. The length of the repeat sequences varies between 25 and 40 nt, whereas the length of the spacer sequences varies between 21 and 72 nt. As mentioned above, some spacers show high homology with foreign nucleic acids, but the origin of a significant percentage of spacers remains unknown. The sequence homology between the spacer and the target is the major determinant of nucleic acid degradation of the target. Some bacterial species contain more than one CRISPR locus within their genome (Louwen et al., 2014). Depending on the specific bacterial species or strain, a CRISPR locus may contain from a few to several hundred repeat spacer units; however, most commonly, a single CRISPR locus contains approximately 50 units. The CRISPR repeat sequences play an important role during both the acquisition of new spacers and the transcription and maturation of CRISPR RNA (crRNA). Based on the sequence similarity, the CRISPR repeats are assigned into groups (Kunin et al., 2007). These groups were taken into account in the current classification of CRISPR-Cas systems (Makarova et al., 2011). Although most CRISPR arrays are located on chromosomal DNA, there are examples of CRISPRs located on plasmids (Godde and Bickerton, 2006). In general, CRISPR loci are flanked by A/T rich leader sequences containing...

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