The discovery of CRISPR/Cas nucleases system
The coexistence of prokaryotes and viruses generated various defense mechanisms such as restriction modifications, toxin-antitoxin systems, and, in the later years, CRISPR/Cas systems were discovered (Labrie et al. 2010). The discovery of CRISPR as a component of the prokaryotic “immune” system, and the repurposing of this system as a genome editing tool, determined a broad use of this technology in molecular biology applications, making it one of the most used technologies in active research in biology (van Soolingen et al. 1993, Bolotin et al. 2005, van der Oost et al. 2009, Mali et al. 2013a, Cho et al. 2013) (Figure 1). Even though the first CRISPRs were observed decades ago in bacteria (Ishino et al. 1987), and subsequent studies revealed the presence of CRISPRs in archaea (Mojica et al. 1993), it was only in the early 2000s that researchers discovered the sequence similarities between the viruses, bacteriophages, and plasmids and the spacer regions in CRISPR, managing to uncover the defense function of CRISPR (Mojica et al. 2005, Bolotin et al. 2005). Independent parallel studies revealed a set of genes associated with CRISPR, consequently named cas (CRISPR-associated), and, in 2008, Marakova et al. suggested the existence of a CRISPR/Cas complex that acts as an acquired immune system to protect the bacterial cell against invading phages or other exogenous genetic material (Jansen et al. 2002, Makarova et al. 2006).
Components of the CRISPR/Cas system
CRISPR/Cas system ensures the immunity of the bacteria in three steps that require target recognition and cleavage (Barrangou and Marraffini 2014, Sorek et al. 2013). The first step is the adaptation, which allows the copy and paste of the foreign nucleic acids into the ‘spacers’ of the CRISPR arrays, thus providing acquired resistance against the invading phage (Barrangou et al. 2007). The next step includes the biogenesis of the crRNA (expression stage), in which the small interfering RNAs are generated through transcription and further processing. In the interference stage, the gRNA heads the Cas enzymes to cleave the DNA (Marraffini and Sontheimer 2008). The CRISPR-Cas system can be classified into two main categories according to the effectors: first, where all functionalities in the effector complexes are carried out using a protein, and second, the multi-unit effector complexes (Shmakov et al. 2017). These two classes are further divided into six types of CRISPR-Cas systems according to the presence of the signature genes. In type I systems, the signature protein is Cas3, in which the cleavage of the external DNA is carried out by the nuclease and helicase domains, and the recognition of the target sequence is made by the multi-protein-crRNA complex Cascade. In type II systems, the unique protein required for the interference is Cas9 (signature protein). In type III systems, the signature gene is cas10, a gene encoding a multidomain protein (Cas10) that is assembled into an interference complex needed for the identification and cleavage of the target sequence. Type IV systems are usually not linked to a CRISPR array. The effector complex is represented by Csf1 (csf1 being considered the signature gene) and the other two proteins encoded by cas genes (Makarova and Koonin 2015). Type V systems comprise a unique Cas9-like nuclease, which can be Cpf1, C2c1, or C2c3 according to the subtype of the CRISPR/Cas type V system (Shmakov et al. 2017, Zetsche et al. 2015). In type VI systems, the protein C2c2 with two HEPN RNase domains is the signature protein (Shmakov et al. 2017). All these systems are classified as either Class1 systems (Type I, III, IV) as they have a multi-subunit effector or as Class 2 systems (Type II, V, VI) as they are characterized by a single-subunit effector (Shmakov et al. 2017, Makarova and Koonin 2015). Table 1 ill...