CRISPR Archives

Six years ago, a new sophisticated prokaryotic defense system, was identified in the structure complex CRISPR-Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR associated nuclease). "Resistance is acquired by incorporating short stretches of invading DNA sequences in genomic CRISPR loci " Brouns, S. J. J., et al. (2008). The CRISPR repeat sequence has been discovered in 1987 but the function remained unknown for the next 20 years. The CRISPR-Cas9 system is an adaptive, heritable defense system that prokaryotes (bacteria and archaea) use to protect themselves by degrading foreign invaders' nucleic acids such as viruses. Recently, these RNA- guided Cas9 nucleases derived from CRISPR/Cas systems have shown promise in transforming our ability to edit mammalian genomes and developing new treatments. I/ Structural characteristics CRISPR consists of an RNA, partly constant with a palindrome, tied to a crRNA sequence DNA specific to a target, and a Cas9 protein (Figure 1). The short guide RNA directs Cas9 to a specific genomic sequence where it induces double-strand breaks that, when imperfectly repaired, yield mutations. Cas9 can also catalyze gene replacement through homologous recombination. A possible additional trans-activating CRISPR RNA (tracrRNA) sequence can be found to help the Cas9-DNA-RNaseIII interaction. Cas9 hosts this RNA guide, place it on the DNA target and cut both strands at the position corresponding to the end of the guide sequence. Figure 1. The CRISPR mechanism of action is simple and modular. Source : Pennisi (2013) II/ Modularity of this complex The simplicity of this system enables the production of RNA targeting very specific DNA sequences in different organisms; Append them to the palindrome and as soon as CRISPR-Cas9 is placed on the DNA, the nuclease will cut the DNA to the specific location. Figure 2. CRISPR-Cas9 complex can be modified for pharmaceutical research by exploiting the CRISPR mechanism of action and changing the Cas9 protein to activate or specifically repress the expression of a gene. Source : Pennisi (2013) We can observe on Figure 2 that the nuclease sites have been removed and the protein receive now either a repressor or an activator sub-unit. It is also possible by modifying the protein to removed the active nuclease sites and attached a protein with known activity on Cas9, in order to associate this new activity to the DNA specificity carried by CRISPR. III/ Turning CRISPR into therapeutics This technology receives a clear interest from the biotech community, three Biotech companies are specialized in the field: Editas (founded by George Church, Zhang and Flagship Ventures), CRISPR Therapeutics (Emmanuel Carpentier, one of those who discovered the Cas9 system, is involved in the company) and Intellia Therapeutics (Cambridge,MA-based Biotech where Jennifer Doudna, third discover, is involved) . Big Pharma companies are getting more and more involved since June 2014. As an example, Novartis announced the 7th of January 2015 that they signed collaboration and licensing agreements with Intellia Therapeutics for the discovery and development of new medicines using CRISPR genome editing technology and Caribou Biosciences for the development of drug discovery tools. Research and development activities will focus on using CRISPR ex vivo for engineering chimeric antigen receptor T-cells (CARTs). Under the terms of the agreement with Intellia, Novartis is receiving exclusive rights to develop all collaboration programs focused on engineered CARTs. CAR-T already relies on genetically manipulating T-cells, programming them to produce antigen receptors that bind to unique cancer cell proteins. CRISPR is an obvious choice of tools to handle this step. And the specific type of stem cells covered in the agreement — hematopoietic stem cells (HSCs), the precursors to blood cells — could be genetically altered to treat hereditary blood disorders, including sickle cell disease and all forms of thalassemia. The main competing technology is TALEN enzymes, widely used in CARTs cell therapies and which showed a massive interest recently. We will publish a new article on this subject soon. Conclusion It has been noted that CRISPR-Cas9 complex sometimes mismatches its target because it can bind to other sites in the Human genome than the desired one. Thus, imperfect Cas9 specificity is a major concern, while several attempts have been made to improve CRISPR specificity, such as using multiple RNA-Cas9 complexes for activity, discovering improved natural Cas orthologs, improved Cas9 variants and judiciously choosing targeting RNAs. And many laboratories are working on optimizing this technology. Nevertheless, it is clear that CRISPR has an enormous potentiel in therapeutic development and many Biotech and Big Pharma companies are already interested. Let the race begin. Reference : The CRISPR Craze, Elizabeth Pennisi, 2013. Science 341 no. 6148 pp. 833-836