A review paper which summarizes the recent findings in the study and characterization of CRISPR-Cas systems in Bacteria and Archaea. Clustered regularly interspaced short palindromic repeats (CRISPR) and Cas (CRISPR associated proteins) is an adaptive defense mechanism present in this primitive organisms, evolved in particular response to persistence stress from biotic elements like viruses and transmissible genetic elements.
Microbes live and evolve in an environment with huge amount of diversity and dynamic environment conditions that are stressful and fluctuating. These stresses can be abiotic (e.g., non-optimal temperature or nutrient levels, redox stress) and biotic (e.g., toxins, viruses, transmissible genetic elements) in nature. The abundance of these factors, especially viruses is a constant threat to the survival of these microbes. This, coupled with the fact that viruses are prone to high rate of mutation and recombinations has led to the development of sophisticated defense mechanism (multilayered, fast-evolving) within the microbial host systems. These systems maintain genetic integrity and co-evolve with the increasing variability and sophistication of their predators; making up for one of the most variable regions within the genome. However, they may occasionally allow exogenous DNA uptake and conservation of genetic material advantageous for adaptation to the environment.
Strategies like prevention of adsoprtion, blocking of injection, abortive infection, restriction-modification system (R-M) and the use of sugar-nonspecific nucleases are often used by hosts to target variety of viral and non-viral attacks.
In this review, however, we examine one recently characterized and studied (although it was discovered long back in 1987) called CRISPR-Cas system (several studies pointed out similarity with RNAi mechanism in eukaryotic systems). CRISPR, as the acronym stands, is a family of DNA repeats found in most archaeal (~90%) and bacterial (~50%) genomes. Cas are CRISPR associated genes that are located upstream of CRISPR loci and code for a family of proteins that carry functional domains typical of nucleases, helicases, polymerases, and polynucleotide-binding proteins.
CRISPR loci typically consists of several noncontiguous direct repeats separated by stretches of variable sequences called spacers (which correspond to segments of captured foreign DNA -- viral and plasmid). Although previous studies were speculative of the role of these spacers, their origin from extrachromosomal elements was established only in 2005 through three independent studies.
These acquired spacer regions essentially act as a immunity repertoire or memory for host cells and allow the effect of any external genetic material containing the exact same spacer sequence, to be cleaved and neutralized using the protein encoded by the Cas genes. A loose analogy to how a mammalian body maintain a repertoire of antibodies (B cells actually) against the infections it has encountered before.
How does it happen?
The repeat-spacer CRISPR array is first transcribed to give a pre-crRNA. The full length pre-crRNA is subsequently processed into specific small RNA molecules that correspond to a spacer flanked by two partial repeats. This processing is achieved with the help of numerous enzymes and complexes depending on the host system (e.g., CASCADE system in E. Coli, Cas6 in Pyrococcus).
The processed crRNA, together with specific Cas proteins, form a CIRSPR ribonucleoprotein (crRNP) complex that facilitates spacr base pairing to the target or matching invasive nucleic acid. The crRNA acts as the guide to allow for the specific base pairing between the exposed crRNA within the complex and the corresponding protospacer on the foreign DNA. The complex then cleaves the foreign DNA thereby neutralizing it.
The whole process thus can be summed up in three stages:
- Immunization, or spacer acquisition, involves the recognition and subsequent integration of spacers between two adjacent repeat units with the CRISPR locus. Spacers appear to be integrate primarily at the leader end of the CRISPR loci.
- CRISPR expression, a primary transcript, or a pre-CRISPR RNA (pre-crRNA) is transcribed from the CRISPR locus by RNA Polymerase. This pre-crRNA is then cleaved into small CRISPR RNAs (crRNAs) by specific endoribonucleases.
- Interference, or immunity, the crRNAs associated (or guide) a multiprotein complex to recognize and base-pair specific regions of incoming foreign DNA (or RNA) that have perfect (or almost perfect) complementarity.
Minute details
While the gist of the process has been described, the paper delves into much detail by describing individual Cas proteins and different types of CRISPR-Cas systems that have been identified depending on how they process the pre-crRNA transcript. Besides, this system has also been observed to play critical role in activity regulation (e.g., DNA repair, gene silencing) and development The mechanism, involvement of complexes varies widely across specifies and any reader is advised to refer the paper for those details.
Towards the end of the review, authors very neatly summarize the discussions and pave way for future biotechnological implications of the CRISPR-Cas system.
- Strain Typing and Epidemiological Studies
- The rapid evolutionary dynamics of CRISPR sequences can provide important insights into the co-evolution of both host and phage populations and allow short, long term analysis by mathematical modeling.
- Development of specific/targeted strains.
- Recent application in the field of highly site specific genome editing by synthesizing custom guide RNAs.
- If CRISPR-Cas system plays so important role, why did almost 50% of the bacterial population didn't acquire it (despite the fact that it is transferable horizontally)?
- The role of the Cas genes need to be better characterized for the whole interplay to become more clear.
- The actual role of PAMs, length of CRISPR loci and the factors which control these need to be explored further.
- How this system could have evolved? What would it look like in even primitive and evolving systems? How did it change over time?
- Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010).
A comprehensive paper with updated facts and proper insight into the process. The language is lucid and comprehensible but I found a few of the sections towards the middle a bit repetitive, since that information was already mentioned and explained in earlier section quite nicely.