CAPÍTULO II: MARCO TEORICO
2.2 BASES TEÓRICAS
2.2.4 La denominada doctrina de la conexión de antijuridicidad
In contrast to eukaryotic genomes, less than 5% of the genome of most
prokaryotic phyla comprises of repetitive DNA (Ussery et al. 2004). Therefore, it has
been proposed that its existence offers certain advantageous characteristics to the carrier, since it survived natural selection over evolutionary time.
A distinct family of direct repetitive DNA sequences in prokaryotic genomes are the clustered regularly interspaced short palindromic repeats (CRISPR). This family was first identified as a distinct class of interspersed short sequence repeats (SSR),
adjacent to the isozyme-converting alkaline phosphatase (iap) gene by Ishino et al. in
1987 and Nakata et al. in 1989 in Escherichia coli K12. The same class of repeats was
found soon in other prokaryotic species such as Mycobacterium tuberculosis
(Hermans et al. 1991), Haloferax mediterranei (Mojica et al. 1995), and Thermotoga
maritima (Nelson et al. 1999). They were recognized as a defined prokaryotic family of
short regularly spaced repeats (SRSR) by Mojica et al. in 2000, who in agreement with
Jansen et al. introduced the acronym CRISPR in an initial study of the CRISPR-
associated system in 2002.
The main, conserved features of the CRISPR system are the following, outlined
in figure 1.5 (reviewed in Sorek et al, 2008; van der Oost et al. 2009; Horvath and
Barrangou, 2010; Karginov and Hannon, 2010; Marraffini and Sontheimer, 2010;
i.
These elements consist of direct repeat sequences which range in size from 21-48 bp (with an average size of 32bp) and in number from 2-375 repeats per locus (with an average of 27). The repeat sequences can be partially palindromic, in the form ofinner and terminal imperfect inverted repeats of up to 11bp (Godde et al. 2006).
CRISPR loci are usually homogenous in their repeat sequence. In terms of sequence conservation, phylogenetically distant species generally show greater variation of the repeat sequences than closely related species, although many
exceptions have been observed (Jansen et al. 2002). The repeats can be divided
into 12 clusters based on sequence similarity and secondary structure formation
(Kunin et al. 2007). Six of these clusters exhibit high and intermediate RNA folding
scores indicating that the repeats, when transcribed, form stable secondary stem- loop structures mediated by the palindromic sequences, hypothetically facilitating recognition by CRISPR-associated proteins. Moreover, some of the clusters contain the conserved sequence GAAA(C/G) in their 3’-terminus, indicating a possible protein binding site. .
ii.
The repeat sequences are regularly spaced by unique intervening sequences(spacers) of variable length, which range in size from 26-72 bp. A fraction of the spacer sequences was found to exhibit significant similarity to sequences from phage DNA and conjugative plasmids, with the highest degree of similarity for a given spacer found within genetic elements associated to the carrier. Taking into account the limited number of characterized viral genomes and conjugative plasmid sequences, it was concluded that the spacer sequences originate from
these foreign genetic elements (Mojica et al. 2005, Pourcel et al. 2005). In support
of this theory, 40% of the spacers in lactic acid bacteria CRISPR loci were found to be homologous to streptococci phage genomes and the respective conjugative
plasmids (Bolotin et al. 2005). Crenarchaeal CRISPR spacers yield matches to
fuselloviruses, rudiviruses and β-lipothrixviruses. Spacer sequence matches were
found in both the sense and anti-sense strands and both gene coding and
intergenic regions of phage genomes (Shah et al. 2009). The viral or plasmid
sequence that is complementary to a given spacer sequence is known as a “protospacer”
iii.
Leader sequences of a size order of 100-550 bp have been detected in associationwith several (but not all) CRISPR loci. They are located directly upstream of the cluster, with respect to the strand orientation of the repeat sequence. These sequences appear to have a high A-T content, are rich in homopolynucleotide regions, lack open reading frames and are generally not conserved between
distantly related species (Jansen et al. 2002), but exhibit similarity between related
species. Analysis of the primary transcripts of CRISPR loci in several species
Lillestol et al. 2006), and putative promoter motifs were identified in leader regions of Sulfolobus acidocaldarius (Lillestol et al. 2006) and E. coli K12 (Pul et al. 2010) confirming that these regions act as transcription promoters for the sense strand of the CRISPR arrays. Moreover, it was initially deduced by comparative analysis
(Lillestol et al. 2006) and subsequently confirmed by genetic studies in
Streptococcus thermophilus (Barrangou et al. 2007) that novel spacers are incorporated along with a novel repeat into the leader proximal end of the CRISPR loci. Therefore, leader regions seem to be playing the dual role of controlling CRISPR transcription and the growth of the array, by interacting with the appropriate proteins for the addition of new spacers.
iv.
A number of protein families have been designated CRISPR-associated (Cas), andtogether with the repeat cluster are regarded as a unified system (Jansen et al.
2002; Haft et al. 2005; Makarova et al. 2006). These families are present only in
CRISPR containing species, located adjacent to the repeat cluster with a generally
conserved orientation. No homologues of the cas genes were found in eukaryotic
or CRISPR-negative genomes. Only one set of cas genes is present in species
carrying multiple CRISPR loci with the same repeat sequence, but if multiple loci
with varied repeat sequence are present, then a respective number of cas gene
sets is observed. A cas gene region can comprise of as many as 20 different,
tandem-arranged genes with no preferential direction of their reading frames, and can be found on either side of a CRISPR locus. An analysis of the Cas genes will be presented in the following section.
Figure 1.5: Graphic representation of a CRISPR locus and the adjacent cas gene operon in a prokaryotic genome
Cas genes are depicted as blue arrows, the leader sequence in red, repeats as dark grey boxes and interspersing spacers as colored, numbered boxes. Direction of transcription is indicated by the black arrow.
The number of CRISPR loci per genome ranges from 0 to 20, with
Methanocaldococcus jannaschii containing the highest number found to date (Jansen
et al. 2002, Godde et al. 2006, Lillestol et al. 2006). According to most recent reviews CRISPR loci are present in 90% of the sequenced archaeal genomes, covering both phyla of Crenarchaeota and Euryarchaeota, and in 40% of the sequenced bacterial
genomes, adding up to 639 genomes analysed up to date (table 1.1, Makarova et al.
CAS genes leader repeat spacer
1 2 3 4
2011a). It has been observed that archaeal clusters, especially from thermophilic organisms, are in general multiple and larger than the bacterial ones, and can represent up to 1% of the prokaryotic genome. Clusters are also present in archaeal
conjugative plasmids, such as pNOB8 and pKEF9 of Sulfolobus sp and bacterial
megaplasmids such as pTT27 of Thermus thermophilus (Lillestol et al. 2006, Godde et
al. 2006). Taxonomic group Genomes analysed Genomes containing cas1 proportion of cas1-containing genomes (%) type I system type II system type III system Archaea Crenarchaeota 17 15 88 15 0 16 Euryarchaeota 47 37 79 33 0 23 Total 67 54 81 50 0 40 Bacteria Actinobacteria 72 26 36 28 15 8 Aquificae 7 5 71 7 1 4 Bacteroidetes- Chlorobi group 32 16 50 14 2 6 Chlamydiae– Verrucomicrobia group 10 2 20 0 1 1 Chloroflexi 10 9 90 9 2 7 Cyanobacteria 14 7 50 7 1 7 Firmicutes 126 56 44 40 17 23 Proteobacteria 318 107 34 117 20 22 Spirochaetes 13 3 23 2 1 0 Thermotogae 11 10 91 10 0 9 Total 639 256 40 245 65 99
Table 1.1: Taxonomic distribution of CRISR-Cas systems in 706 analysed genomes
Different CRISPR system types can coexist in different genomes. Adapted from Makarova et al. 2011a.
The origin of spacer sequences and the analogies observed by Makarova et al.
(2006) between the system components and the eukaryotic RNA interference led a number of groups to propose that the CRISPR loci and their associated genes represent an adaptive prokaryotic resistance system against infections from
extrachromosomal elements, functioning via RNA interference (Mojica et al. 2005,
(2005) had already combined reports of foreign genetic elements such as viruses and conjugative plasmids failing to infect CRISPR-carrier strains whose spacers exhibited homology with these elements. The first experimental validation of the CRISPR
function was achieved in 2007 by Barrangou et al. when CRISPR loci of
Streptococcus thermophilus were shown to incorporate new spacers homologous to phage genomic sequences during the generation of phage-resistant mutants, and resistance specificity was shown to depend on the spacer sequence content. In the
same study, knockout of two cas genes resulted in loss of phage resistance and
inability to incorporate new spacers respectively, confirming the functional association
of the cas genes with the CRISPR elements (Barrangou et al. 2007).
Figure 1.6: Outline of the CRISPR/Cas mode of action
The current model for the three stages (adaptation, expression and interference) of CRISPR functioning for each subtype, as inferred by genetic and biochemical studies discussed in this chapter (adapted from Makarova et al. 2011a).
According to the proposed mechanism (Makarova et al. 2006), the entire CRISPR repeat region is theoretically transcribed as a single primary transcript, and after a series of processing steps small interfering antisense RNA molecules of the size of a repeat/spacer unit (referred to as mature crRNAs or psiRNAs in the literature) are produced. This procedure could be under regulation by Cas proteins and induced by stress or phage infection. The mature psiRNA molecules could then anneal to the respective foreign mRNA, resulting in translation repression or cleavage of the dsRNA molecule, thus silencing the foreign genes and inhibiting phage or plasmid proliferation (figure 1.6). The Cas proteins are proposed to comprise the protein machinery of this immune system, mediating the generation and maintenance of the CRISPR loci, the processing and integration of new spacers as well as the RNA silencing process. The discrete stages of this mechanism will be discussed in more detail in subsequent sections.