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CPISPR Applications

By , MD, PhD

Regularly clustered, interspaced, short palindromic repeats (CRISPR) represents a family of DNA repeats initially described in 1987 in an intergenic region in the E. coli K12 genome, which are often accompanied by CRISPR-associated proteins (Cas). The wide variety of CRISPR sequences found in the microbial world offers an opportunity to exploit them in various applications.

The primary applications of CRISPR systems include the use of these arrays’ hypervariability for genotyping purposes (primarily for pathogenic strains) and crRNA-mediated interference for phage resistance in industrially relevant strains. In recent years, precise manipulation of the genome is one of the revolutionary applications in biomedical research endeavors.

Genotyping

CRISPR loci and its diversity were initially exploited to type Mycobacterium and Yersinia strains, which is also known as spacer-oligotyping technique (spoligotyping). The principle of this easy-to-perform method is PCR amplification of the entire CRISPR locus with primers that are labeled and that recognize the Direct Repeat (DR) sequence. As the spacer content is strain specific, differential hybridization patterns enable separation of the strains.

With several improvements to the classical spoligotyping (such as automation and use of microbeads instead of a membrane), this technique remains a gold standard for subtyping of members of Mycobacterium tuberculosis complex. Next generation spoliogtyping is also used for typing Salmonella and Campylobacter jejuni strains.

CRISPR loci provide a way to probe different ecological populations in order to resolve the diversity of host and viral populations in compound systems – for example, in natural habitats and human samples. The use of metagenomic deep sequencing techniques for extensive analysis of CRISPR sequences can give paramount insights into the evolutionary trajectories and co-dynamics of both host and virus populations.

Phage-resistance

From an industrial point of view, where bacteriophage attack is recognized as a major problem in dairy fermentative processes for almost 80 years, there is a recognized need for transfer of phage-resistance systems to phage-sensitive strains of industrial importance.

Among the different approaches used to generate phage-resistant strains is the exploitation of CRISPR/Cas systems. Protocols of selecting CRISPR-containing strains without deliberate genetic modifications are already in place for dairy Streptococcus thermophilus. Conversely, another possibility of constructing phage-resistant strains is the integration of synthetic spacers that match conserved sequences of industrially occurring phages into the CRISPR array of starter bacteria.

The benefits of using CRISPR-based variants are large and very promissory. In particular, the fermentative industry can be benefited greatly by the inclusion of phage-resistant variants to the pool of starting cultures. Furthermore, CRISPR-mediated protection against conjugative plasmids can be used to limit the spread of antibiotic-resistant strains in hospitals.

Genome Engineering

CRISPR/Cas technology has undoubtedly revolutionized editing of the genome, granting a hitherto unachievable level of genomic targeting, simplicity and efficiency. Laboratories worldwide are currently using this technology for unprecedented applications in biomedicine.

Although type I and III CRISPR/Cas systems show RNA-guided nuclease activity, it is carried out in large and multimeric ribonucleoprotein complexes, which hampers its development as a molecular tool. Conversely, type II system relies only on a single endonuclease (most notably Cas9) which can generate anticipatable breaks in the target sequence.

The ease of CRISPR/Cas9 programming (in sync with a unique DNA cleaving mechanism), multiplexed recognition of the target, as well as a panoply of type II CRISPR/Cas system variants in nature, have led to exceptional developments using this relatively simple and cost-effective technology to edit, regulate, modify and mark genomic loci of a myriad of organisms.

Sources

  1. http://aem.asm.org/content/80/2/430.full
  2. http://genesdev.cshlp.org/content/28/17/1859.full
  3. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/
  4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433013/
  5. http://www.sciencedirect.com/science/article/pii/S0300908415001042
  6. http://biochem158.stanford.edu/Final%20Papers%202014/Dubrow%202014.pdf
  7. Martínez B, García P, Rodríguez A, Piuri M, Raya RR. Bacteriophages of Lactic Acid Bacteria and Biotechnological Tools. In: Mozzi F, Raya RR, Vignolo GM, editors. Biotechnology of Lactic Acid Bacteria: Novel Applications. John Wiley & Sons, Ltd, 2016; pp. 100-119

Further Reading

Last Updated: Jan 13, 2016

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