The recently developed CRISPR gene editing technology is currently generating significant interest in the biomedical community, not least of all due to its low cost, its incredible ease of use and its versatility across a broad range of applications (see Bayer-CRISPR Therapeutics joint venture announcement in this issue). Referred to as “clustered regularly interspaced short palindromic repeats” or “CRISPR/Cas9”, the technology has emerged from research into prokaryotic immune defense systems. However, the mechanisms at play have recently been shown to correct congenital cataract in mammalian models, with many additional applications in the pipeline. Remarkably, this very ancient bacterial technology is being rapidly applied to several areas of 21st century medicine and biotechnology. The impact is additionally been felt beyond the university walls with >$150M of venture capital invested to date in 4 start-up companies alone. Such rapid commercial interest in a technology with 10+ years to go to reach market is a clear indicator of the size of CRISPR’s market opportunities.
Independent of the general euphoria around the technology, CRISPR/Cas9 is a “big” development within biotechnology. The core opportunity has been developed by a number of key players, chiefly the founding scientists currently associated with several start-up companies including Crispr Therapeutics (Basel, Switzerland), Editas Medicine (Cambridge, Massachusetts), Caribou Biosciences, Inc. (Berkeley, California), Intellia Therapeutics (Cambridge, Massachusetts), and ERS Genomics (Dublin, Ireland).
The technology derives from observations made in the repetitive sequences isolated from a number of prokaryotic and archaebacteria, first identified in 1987 by Yoshizumi Ishino and Atsuo Nakata, then at Japan’s Research Institute for Microbial Diseases, Osaka University. Working on the E.coli iap enzyme, responsible for the isozyme conversion of alkaline phosphatase, Ishino and colleagues reported an unusual set of 29 nucleotide repeats interspersed with five intervening 32 nucleotide sequences, seemingly without any discernible function. Over a period of 10+ years, as increasing numbers of DNA sequences were deposited in the public databases, a growing number of similarly structured repeat sequences, interspersed with unusual sequence, were reported from several different bacterial and archael strains.
By 2002, Jansen and colleagues at the Department of Infectious Diseases and Immunology, Utrecht University, used in silico analysis to characterize such families of repetitive DNA sequences within archaea and bacteria but absent from eukaryotes and viruses. They coined the term “clustered regularly interspaced short palindromic repeats” (CRISPR), although interest in the field remained relatively non-existent.
The spark that lit the fuse came in three papers in 2005 when Francisco Mojica and Elena Soria at the Univerisidad de Alicante in Spain; Pourcel, Salvignol and Vergnaud at the Universite Paris XI, Orsay, France, and; Alexander Bolotin and Stanislav Dusko Ehrlich at the Institut National de la Recherche Agronomique, also in France, reported that the spacer sequences separating the individual direct repeats appeared to have a phage associated origin. Coupled to this were separate observations that viruses were unable to infect cells that carried spacer sequences corresponding to their own genomes. In essence, the system as a whole appeared to represent an un-expected and sophisticated immune system for prokaryotes, essentially a new mechanism that provided an immune memory of previous phage infections and facilitated rapid clearing of subsequent phage invasions that had previously infected the cell. Research progressed rapidly and by 2012 a paper published in Science by Jinek and colleagues at the University of California, Berkeley, in collaboration with the University of Vienna and Umeå University in Sweden, harnessed the technology into a system that is “efficient, versatile, and programmable” with “considerable potential for gene-targeting and genome-editing applications.” Further iterations and development showed how relatively easy the CRISPR/Cas9 system could be used as an RNA-guided platform for specific control of gene expression. When compared to other editing tools, such as zinc finger proteins or TALENs, CRISPR/Cas9 has proved to be remarkably cheaper and less time consuming to work with.
In 2013, researchers at the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, used CRISPR/Cas9 technology to correct a dominant mutation in the mouse crystallin gamma C gene (Crygc), mutations of which may cause significant cataracts. Injection of Cas9 mRNA and a single-guide RNA (sgRNA) targeting the mutant Crygc allele, into animal zygotes, facilitated gene correction via homology-directed repair based on either a supplied oligo or the wild type allele. The researchers reported minimal off-target modifications and confirmed that treated animals transmitted the corrected allele to their progeny.
In the field of CRISPR, with applications varying from dairy industry starter-cultures to crop sciences to neurological gene therapy and stem cell biology, it may be somewhat of an under-statement to suggest that CRISPR is a very big development. More critically, the field of ophthalmology may be one of the early beneficiaries of what has become a truly remarkable field within the cell and gene therapy market.