Genetic Engineering: Evolution and Revolution

Over the last years, new genetic engineering technologies have emerged and undoubtedly show up game-changing. Genome engineering is since a long time explored and used because there is a lot at stake here in many fields. Indeed, the possibility to add, remove or change precisely DNA sequences in many cell lines or organisms have great involvements for fundamental research or therapeutic applications.

At the beginning, scientists have taken recombinases or integrases existing in nature to undertake specific genetic changes, but these strategies are limited by the wild specificity of these proteins. Then, scientists tried to modify these enzymes to move from the specificity to the targeting DNA modifications. After several round of mutagenesis, different integrases or recombinases have been generated to target new chosen loci but the efficiency was low and the off targeting high.

In 2005, scientists designed an artificial class of genome editing tools by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain and called Zinc Finger Nuclease (ZFN)(Urnov et al., 2005). It was the first time that scientists achieved to generate an efficient and specific synthetic genome editing tool. This success has been initiated thanks to research undertaken by Aaron Klug at universities of London and Cambridge for his structural elucidation of biologically important nucleic acid-protein complexes and for which he won the Nobel price (Klug and Rhodes, 1987).

ZFN is a very powerful tool because it allows targeting desired DNA sequences by engineering the zinc finger domains according the needs. This class of genome editing tools shows a higher efficiency, specificity and flexibility than modified recombinases although off target events are also observed. However this is not the biggest problem, the real lock of use is the tricky design of ZFNs combined to the IP landscape. Only few labs are able to design ZFN and Sangamo which is patent owner of the technology locks the domain with prohibitive prices (Scott, 2005). Consequently very few labs have access to this attractive technology, limiting its use and the discoveries done with.    

In 2009 a new class of artificial genome editing tool emerged, the Transcription Activator-Like Effector Nucleases (TALEN) which is made by fusing TAL effector DNA binding domain to a DNA cleavage domain (Moscou and Bogdanove, 2009). Overall the scientific publications shown that TALENs have a similar efficiency and specificity than ZFNs. Principal difference is about the complexity of engineering, TALENs are much simple to design because there is a straightforward relationship between amino acid sequence and DNA recognition. Consequently, many labs are able to design and produce its own TALENs for their research projects. Nevertheless, TALENs are very large proteins with many repeat sequences generating a long timing and high cost of production. Furthermore, these features limit the efficiency of TALEN delivery in several experimental contexts.

Few years later, in 2012, a new synthetic genome editing tools is designed jointly by US and European labs: the CRISPR-Cas9 system (Cong et al., 2013; Jinek et al., 2012). This system is initially a prokaryotic immune system which is composed of RNA guides helping a Cas protein to recognize and cut exogenous DNA. This system has been modified and adapted to works efficiently in eukaryotic cells in vitro and in vivo. CRISPR-Cas9 is very easy to design, cheap and fast to produce. Consequently, this tool has democratized the use of genome editing tools, indeed all labs in molecular biology are able to design their own tools with low cost for genome editing. It is why CRISPR-Cas9 has triggered a revolution in which laboratories around the world are using the technology for innovative applications in biology. Scientific publications constitute a very strong indicator about that, before 2012 you find around 200 publications about CRISPR, and after 2012 more than 5.000.

CRISPR use open new avenues in a wide range of applications, as well as for the gene studies and mechanisms in fundamental research than for therapeutic fields in cell and gene therapy. Furthermore, recent modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. These new designs made of CRISPR-Cas9 an unprecedented toolbox for genome and epigenome editing. However, the use of this promising tool is limited in several contexts, mostly for therapeutic applications, because of low efficient delivery. To overcome this limit, several recent studies shown the possibility to vectorize the CRISPR-Cas9 system in efficient viral vectors. By the way, recent works demonstrated the power of the CRISPR-Cas9 vectorization in lentiviral vector to generate CAR T-cells (CAR: Chimeric Antigen Receptor) which seems very promising to design new strategies against cancer.

In February 2017, first issues about the landscape of CRISPR-Cas9 system have been resolved and companies which made business of it have to be careful about that. However, for all non-commercials use which represents many cases, any restriction existing, the CRISPR revolution is underway!

Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et al. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339, 819–823.

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier, E. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337, 816–821.

Klug, A., and Rhodes, D. (1987). Zinc Fingers: A Novel Protein Fold for Nucleic Acid Recognition. Cold Spring Harb. Symp. Quant. Biol. 52, 473–482.

Moscou, M.J., and Bogdanove, A.J. (2009). A Simple Cipher Governs DNA Recognition by TAL Effectors. Science 326, 1501–1501.

Scott, C.T. (2005). The zinc finger nuclease monopoly. Nat. Biotechnol. 23, 915–918.

Urnov, F.D., Miller, J.C., Lee, Y.-L., Beausejour, C.M., Rock, J.M., Augustus, S., Jamieson, A.C., Porteus, M.H., Gregory, P.D., and Holmes, M.C. (2005). Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651.

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