The CRISPR/Cas9 system allows a precise region of DNA to be modified quickly and cheaply and has captured the attention of the entire biotechnological community due to its versatility and its countless applications. However, the advent of CRISPR/Cas9 opens up many new questions of a bioethical nature.
From natural immune mechanism to powerful biotechnological tool
To understand the functioning of the genes of our body and of any organism, it is important to study what happens when its expression is modified. For centuries, scientists had to be content with observing the effects of spontaneous mutations or using substances that introduced random errors into the genome of plants, animals or cultured cells. In recent years, new techniques have been developed, called “genome editing”, which allow precise modifications to be made to the genes to be studied, greatly speeding up progress in biological research. In the future, these techniques could also be used to “correct” alterations responsible for genetic diseases or modify some organism characteristics. Among them, the most recent and promising system is known as CRISPR/Cas9.
Behind this name originally lies an immune mechanism that some single-celled organisms (such as bacteria) use to defend themselves against viruses. These organisms contain fragments of “guide” RNA known as CRISPR, which function as molecular sentinels by recognizing foreign DNA sequences by base pairing. Once the foreign DNA is recognized and latched onto, CRISPR guides an enzyme onto it called Cas9 (CRISPR-associated), an endonuclease which, functioning like a pair of scissors, cuts the intruding DNA, preventing its replication. The CRISPR sequences were already discovered in 1987 by Ishino and colleagues, but their role was completely understood only thirty years later, in 2007.
Since then and in particular since, in 2012, the system was made customizable by the research groups of Jennifer Doudna and Emmanuelle Charpentier, biotechnologists have been trying to exploit this natural defense system to introduce specific modifications in the genome of organisms much more complex than bacteria, such as animals and plants. To do this, the CRISPR system is used coupled to the DNA repair systems present in the cell, which, after being cut by Cas, operate a mending according to different mechanisms. Some of these mechanisms are inherently imprecise and introduce “errors” into the sequence, so that sometimes the modified gene becomes no longer functional. This modality can be exploited to eliminate the expression of a gene whose function is to be studied. Another repair mechanism, called “homologous recombination”, acts with precision, allowing the cut to be repaired and the desired sequences to be inserted. Thanks to this strategy, it is possible not only to study the effect of introducing certain mutations but also, for example, to correct mutations that cause genetic diseases.
The system has already been used successfully to resolve long-standing scientific controversies [5] and to understand the role of many genes with unknown function [6]. It is a real revolution in the biotechnological field, which has aroused growing interest, as also demonstrated by the rapid surge in publications: if the first scientific article proposing the use of CRISPR for genetic engineering was published in 2012 , today the Pubmed scientific portal already has thousands of bibliographic references on the subject, including original scientific articles and reviews.
The CRISPR/Cas system is also gradually supplanting more obsolete methods for studying the genetic causes and course of diseases, such as various types of cancer (on cell and animal models), as well as the efficacy of drugs. One of the most promising research field is disease of the immune system, where cells can be isolated, modified in vitro and reintroduced into the patient once “corrected”. This strategy has already made it possible to generate HIV-resistant T lymphocytes in vitro.
In the plant sector, the CRISPR/Cas system shows surprising potential. Not only does it prove to be an important tool for basic research, but it also allows the generation of plants with better nutritional characteristics or resistant to pathogens, as in the case of a recent variety of tomato resistant to powdery mildew being perfected in the laboratories of the Sainsbury Laboratory in Norwich (Great Britain). Although all of this is already partially possible with traditional crosses, the CRISPR/Cas system is much more specific and allows results to be obtained in a few months rather than years.
Furthermore, compared to the molecular biology techniques used up to now, it does not require the introduction of portions of DNA from other species, such as genes for resistance to antibiotics or herbicides. The Cas protein itself is introduced only for the time necessary to correct the plant genome, but can subsequently be eliminated without a trace, thanks to targeted crossings with the wild plant.
References
Ishino, Yoshizumi, et al. “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 (1987): 5429-5433.
Barrangou, Rodolphe, et al. “CRISPR provides acquired resistance against viruses in prokaryotes.” Science 315.5819 (2007): 1709-1712.
Jinek, Martin, et al. “A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity” Science, 337.6096 (2012), 816-821
Gilbert, Luke A., et al. “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.” Cell 154.2 (2013): 442-451.
Gao, Yangbin, et al. “Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development.” Proceedings of the National Academy of Sciences 112.7 (2015): 2275-2280.
Swiech, Lukasz, et al. “In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9.” Nature biotechnology 33.1 (2015): 102-106.
Sánchez-Rivera, Francisco J., and Tyler Jacks. “Applications of the CRISPR-Cas9 system in cancer biology.” Nature Reviews Cancer (2015).
Schumann, Kathrin, et al. “Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.” Proceedings of the National Academy of Sciences (2015): 201512503.
Vladimir Nekrasov. Tomelo- rapid generation of a tomato resistant to the powdery mildew by genome editing. CRISPR/Cas Workshop, 7-8 September 2015, John Innes Centre, Norwich (GB).
Belhaj, Khaoula, et al. “Editing plant genomes with CRISPR/Cas9.” Current opinion in biotechnology 32 (2015): 76-84.
