CRISPR under the microscope –
A Bacterial defence system
This is designed to be an in-depth article for those with some knowledge of CRISPR, if you would like a simple overview or a new to the topic please read The Foundations of CRISPR first.
CRISPR array refers to the region of the genome that contains the genes associated with CRISPR functioning. This unusual genomic feature was first noticed in Escherichia coli by Ishino et al. (1987) and since then, has been found widely across prokaryotic genomes. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats (Jansen et al., 2002), a description of the appearance of the array in the genome reflecting that the feature was observed before the purpose and role of the CRISPR array in bacterial defence was understood.
Structure of the CRISPR array
There are several features in the CRSIPR array. First there is the leader sequence which is responsible for the transcription of the array, this is followed by Cas proteins, CRISPR associated proteins (Charpentier et al., 2015). Different species contain Cas proteins of different structures but they generally fulfil a similar role with the defence system. The Cas protein genes are followed by the Spacer and Repeat sections. The repeat sections are the palindromic sections referred to in the name CRISPR, whereas the spacer sections are unique. Matching these spacer sections to the genome of known phages (viruses that target bacteria) was the first evidence that CRISPR had a role in bacterial defence (Bolotin et al., 2005; Mojica et al, 2005; Pourcel et al., 2005).
Expression of the CRISPR array
In response to the presence of the invasive genetic material of phages the leader sequence initiates the transcription of the CRISPR array (Charpentier et al., 2015). The Cas genes are translated into Cas proteins but remain inactive in the cytosol until activated by the products of the spacer and repeat transcription. As the spacer and repeat sections are transcribed the repeats sections form r-loops, base pairing to form a hairpin turn, thus creating pre-crRNA. The R-loops are important for the processing of the spacers and repeats from pre-crRNA into crRNA. The processing is performed by either the Cas protein or tracrRNA (trans-activating crRNA) depending on the Cas proteins within the cell. The resulting crRNA contains both a spacer and partial repeat which can binds to the cytosolic Cas proteins thus forming an activated crRNA/Cas complex.
Targeting of invasive DNA
The binding of the crRNA to the Cas protein not only activates the Cas protein but also provides the mechanism through which the invasive DNA is targeted. The spacer within the crRNA targets the mechanism towards the DNA. It contains a 20 bp sequence that complementary base pairs with invasive DNA (Gasiunas et al., 2012; Jineket al., 2012).
In order to prevent the crRNA/Cas complex from binding to the cell’s own DNA the Cas protein contains a PAM (proto-spacer adjacent motif) site. PAM is a short nucleotide sequence that is recognised by the crRNA/Cas complex and is not present in the organism’s own DNA. Thus, the presence of a PAM site means that the cell can determine self from non-self (Marraffini and Sontheimer, 2010). There are different PAM sites for different Cas proteins or the same Cas proteins if they originate from different organisms. (Mahas et al., 2017).
Cutting the invasive DNA
When the Cas/crRNA complex is bound to the DNA it brings the nucleolytic domains of the Cas protein into contact with the DNA. These domains cleave the backbone of the DNA creating the double strand break. Different Cas proteins have different domains for this purpose. CRISPR systems are divided into classes types based on this diversity (Ishino, 2018). In the much-used Cas9, the domains are HNH and RuvC domains, which respectively cleave the complementary and non-complementary DNA strands (Sapranauskas et al., 2011). Through cleaving the invasive DNA multiple times, it leaves the viral DNA unable to hijack the cell’s machinery and vulnerable to degradation by powerful cytosolic nucleases.
Integrating DNA into CRISPR array
The system described thus far has a spacer within the CRISPR array that corresponds to the invading phage DNA. However, this cannot always be the case as there must an initial infection. The bacterial cell contains other methods of targeting the invasive DNA, including endonucleases which degrade the DNA (Seed, 2015). This leaves behind short fragments of the phage DNA which can be added to the CRISPR array in what is termed the acquisition phase (Ishino. 2018). This forms an adaptive immunity for the bacterium. An immunological memory is created with the CRISPR array of previous invasive DNA, thereby providing a targeted immune approach to any subsequent infections by the same viral DNA.
Summary
The CRISPR system is a mechanism through which bacteria can defend itself against phages. Stored in the CRISPR array are fragments of previous invasive DNA infections which are expressed when a new infection occurs. These fragments are then used to target any corresponding DNA for degradation, thereby eliminating the threat. Fragments of phage DNA in the cell are incorporated into the CRISPR array and stored for later use. Thus creating a system of adaptive bacterial immunity.
Learn More from Talking Biology
The Foundations of Genetic Editing – CRISPR
This covers both CRISPR as a bacterial defence system and its repurposing into a widely used genetic editing technique.
An overview of how and why you would genetically engineer an organism.
References
Bolotin, A. et al. (2005) ‘Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin’, Microbiology, 151(8), pp. 2551–2561. doi: 10.1099/mic.0.28048-0.
Charpentier, E. et al. (2015) ‘Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity’, FEMS Microbiology Reviews, 39(3), pp. 428–441. doi: 10.1093/femsre/fuv023.
Gasiunas, G. et al. (2012) ‘Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria’, Proceedings of the National Academy of Sciences, 109(39), pp. 2579–2586. doi: 10.1073/pnas.1208507109.
Ishino, Y. et al. (1987) ‘Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product.’, Journal of Bacteriology, 169(12), pp. 5429–5433. doi: 10.1128/jb.169.12.5429-5433.1987.
Ishino, Y., Krupovic, M. and Forterre, P. (2018) ‘History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology’, Journal of Bacteriology, 200(7), pp. 1–17.
Jansen, R. et al. (2002) ‘Identification of genes that are associated with DNA repeats in prokaryotes’, Molecular Microbiology, 43(6), pp. 1565–1575. doi: 10.1046/j.1365-2958.2002.02839.x.
Jinek, M. et al. (2012) ‘A Programmable Dual-RNA – Guided DNA Endonuclease in Adaptive Bacterial Immunity’, Science, 337(August), pp. 816–822. doi: 10.1126/science.1225829.
Mahas, A., Neal Stewart, C. and Mahfouz, M. M. (2017) ‘Harnessing CRISPR/Cas systems for programmable transcriptional and post-transcriptional regulation’, Biotechnology Advances. Elsevier, 36(1), pp. 295–310. doi: 10.1016/j.biotechadv.2017.11.008.
Marraffini, L. A. and Sontheimer, E. J. (2010) ‘Self vs. non-self discrimination during CRISPR RNA-directed immunity’, Molecular Biology, 463(7280), pp. 568–571. doi: 10.1038/nature08703.Self.
Mojica, F. J. M. et al. (2005) ‘Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements’, Journal of Molecular Evolution, 60(2), pp. 174–182. doi: 10.1007/s00239-004-0046-3.
Pourcel, C., Salvignol, G. and Vergnaud, G. (2005) ‘CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies’, Microbiology, 151(3), pp. 653–663. doi: 10.1099/mic.0.27437-0.
Sapranauskas, R. et al. (2011) ‘The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli’, Nucleic Acids Research, 39(21), pp. 9275–9282. doi: 10.1093/nar/gkr606.
Seed, K. D. (2015) ‘Battling Phages: How Bacteria Defend against Viral Attack’, PLoS Pathogens, 11(6), pp. 1–5. doi: 10.1371/journal.ppat.1004847.
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