Is based on 1987 noted that 29 nucleotide repeat sequences in the E. coli genome were sequenced at 32 nucleotide intervals, investigating how alkaline phosphatase isoenzyme transformations were performed in Escherichia coli.
In the following process, a number of such similar repeat sequences have been identified in different bacteria and backgrounds. According to Mojica et al. These short repeat sequences are present in about 40% of the bacteria and 90% of the archaea.
These short sequences were identified as (Clustered Regularly Interspaced Short Palindromic Repeat) in 2002, and since then they have been shortly called CRISPR. Later clustering of Cas genes near the CRISPR regions was found in prokaryotes .
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and related proteins (Cas), which make up the CRISPR-Cas system, provide adaptive immunity against foreign substances in many bacteria and many Archaea.
CRISPR-Cas systems include CRISPR-related (Cas) genes and corresponding CRISPR sequences.
For CRISPR activity, the presence of CRISPR related (Cas) genes adjacent to CRISPR sequences and encoding proteins for immunological response are needed.
These characteristic CRISPR sequences are formed by the insertion of non-repeated sequences derived from short segments of foreign genetic material into repetitive sequences.
That is, certain parts of the bacterium-infecting virus DNA are re-inserted into the CRISPR region along with their genes. Another feature associated with CRISPR regions is the presence of conserved sequences, called leaders, behind CRISPR according to the transcription direction.
Although the presence of these leaders sequences was initially observed only in Methanocaldococcus jannaschii, Archaeoglobus fulgidus and Methanothermobacter thermautotrophicus, it was found in many species of bacteria in later studies.
The sequence and length of repeat regions and the length of the interval regions are well preserved in the CRISPR region, but these CRISPR regions may differ in the same or different genomes. The repeat sequences are between 21-48 base pairs (bp) while the spacing ranges between 26-72 bp. The spacing in the CRISPR region varies widely from several to several hundred.
The genome may contain single or multiple CRISPR regions and, in some species, these regions may constitute an important part of the chromosome. For example, Methanocaldococcus sp. The CRISPR regions in FS406-22 (including 18 CRISPR and 191 spacing) and Sulfolobus tokodaii 7 (containing five CRISPR and 458 spacing) constitute 1% of the genome.
The CRISPR-Cas immune system performs immunity in the cell in three steps (Figure 1.2). The first step is an adaptation in which the cleavage fractions obtained from the exogenous nucleic acid are placed in the CRISPR region(Barrangou and Marraffini 2014). In this step, the selection of proto intervals is determined by the specific recognition of the protospacer adjacent motif-PAM in the invading plasmid and phage genomes(Jiang and Doudna 2015). PAMs are highly conserved sequence motifs composed of 2-5 nucleotides.
The foreign DNA spacer portion with the PAM sequence is inserted into the CRISPR region along with the repetitive genes. Since the lack of the PAM recognition sequences in the repetitions of the bacterial CRISPR region precludes the possibility of self-targeting and self-splicing of the CRISPR-Cas systems, Mutations in the PAM sequence allow the phage to escape the CRISPR immunity.
In the second step, the region inserted into the CRISPR locus of the target sequence in the invasive DNA is transcribed into precursor CRISPR RNAs (pre-crRNA) and the resulting pre-crRNA transcripts are converted into small crRNAs that express Watson-Crick base pairing with exogenous DNA target sequences by Cas endoribonuclease (The sequence of the crRNA corresponds to the sequence of the invading DNA).
The final step is targeting, targeting invasive nucleic acids using crRNA and Watson-Crick base pairing, and by cutting homologous sequences with Cas nuclease to prevent the proliferation of viruses and plasmids.
Although many CRISPR loci were identified afterwards, the biological significance of this was not understood until 2005. At this time, three independent researchers identified CRISPR as containing phage and plasmid-based spacer sequences.
As a result of further studies, it was concluded in 2007 that CRISPR is actually an adaptive immune response mechanism that protects against the bacterial genome, phage and plasmids.
Subsequent studies have revealed that CRISPR has a detailed structure that will actually form a system, by identifying Cas gene, Cas protein, PAM motif (protospacer adjacent motif), crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA).
Although all aspects of the CRISPR system have not yet been fully elucidated, the general functions and processes of formation have been determined in a significant way.
CRISPR-Case System Types
Cas proteins are very diverse and interact with nucleic acids such as nucleases, helicases and RNA binding proteins. Cas1 and Cas2 proteins play a role in adaptation and these proteins are present in all CRISPR-Cas systems. Other Cas proteins are only associated with certain types of CRISPR-Cas systems.
The diversity of Cas proteins, the presence of multiple CRISPR regions, and the transition between living beings make it difficult to classify CRISPR-Cas systems. According to the organization of the CRISPR region and the content of the Cas genes, CRISPR-Cas systems are classified into three main types(I, II and III) and 11 subtypes(I-A to I-F, II-A to II-C, III-A to III-B).
The CRISPR system has been divided into three categories as type I, type II and type III according to homology between Cas proteins. While only one Cas protein is sufficient for the identification and segmentation of target regions of Type II, Cas protein sets are required for Type I and III to function.
Since the Cas proteins are responsible for the biogenesis of crRNA and recognition and degradation of invasive nucleic acids, the molecular mechanism of each CRISPR type is specific(Barrangou and Marraffini 2014).
In Type I and Type III systems, the endoribonucleolytic cleavage of the repeated sequences of pre-crRNAs to produce small mature crRNAs are based on the Cas6 nuclease family(Jiang and Doudna 2015). In the Type I system, the resulting crRNA molecule combines with Cascade and Cas3 proteins to cut foreign DNA, but In the type III system, the crRNA that forming from pre-crRNA is complexed with Cmr / Cas10 or Csm / Cas10 proteins, and the complex Cas proteins cut foreign DNA.
The Type II system is the system that works best among these systems and is the best-illuminated system. In the type II system, a ribonucleoprotein complex with a non-coding RNA (trans-activating CRISPR RNA (tracrRNA)), crRNA, endonuclease Cas9 is complexed and recognizes and intercepts invasive DNA.
There may be different types of CRISPR-Cas systems in a single organism. The CRISPR-Cas systems are given the crRNA biogenesis and targeting mechanisms.
CRISPR type II properties have made it a target for genome editing studies. The presence of Cas9-crRNA complexes, which function as RNA-guided endonucleases in Streptococcus thermophilus and Streptococcus pyogenes, in 2012, has also led to the potential existence of the CRISPR system for genome editing.
Cas9-crRNA constitutes a complex of Guide RNA that recognizes target sequences with the Cas9 protein that cuts DNA from specific regions.
All these Data suggest that the Cas9-crRNA complex may be a valuable tool for genome editing. The fact that the CRISPR / Cas9 system is open to modification has opened up new approaches and this technology has been accepted as a new era in targeted technologies.