The ability to manipulate the genome of bacteria and viruses is now a common tool of experimental biology. More recently, technologies have emerged that allow the development of genetically manipulated animals for use in research and commercial applications. However, until recently, production of genetically manipulated animals was an expensive and laborious process that relied on spontaneous homologous recombination in embryonic stem cells. It was not unusual for the cost of this technology to exceed $20,000 and the process to take over 12 months to complete.
In the past few years, technologies for directing the targeting of mutations to specific sequences and rapidly incorporating novel DNA into the genome have been developed and are revolutionizing our ability to manipulate and study genes. Among these are zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-9 system. Of the three, CRISPR-Cas9 technology shows the most potential and has generated the most interest (1). The system was first identified as a mechanism for adaptive immunity in eubacteria and archaea. It was discovered that some bacteria store short DNA sequences from the genome of viruses that have previously invaded a cell or its ancestors and use these sequences to inactivate similar pathogens that are attempting to attack the cell. However, CRISPR-Cas9 is one of those discoveries of pure basic research that is now found to have wide-reaching implications. In the last two years it has been demonstrated that this system could be easily used to manipulate mammalian genetic material in numerous species.
Cas9, which stands for CRISPR associated protein 9, is an RNA-guided DNA nuclease enzyme. It cuts DNA at a site that is directed by “guide RNA”. In bacteria this guide RNA is transcribed from the stored short pathogen sequences in the bacterial genome that are the result of previous infection. However, for genetic engineering the guide RNA is generated artificially. The elegance of the system is its high specificity for introducing double stranded cuts in DNA. Co-introduction of targeting vectors, which contain DNA sequences that direct cells to incorporate specific new DNA sequences into the genome at the Cas-9-cut site, facilitates highly efficient, targeted DNA manipulation.
Recent use of this technology has seen success in generating both “knock out” and “floxed” mice (2) for use in defining the in vivo function of genes with global or targeted gene deletion. The theoretical advantages of creating animals using this technology over other available methods are the ease of preparing specific reagents and the significant decrease in associated costs and time for generating animals. The technology also permits the introduction of genetic mutations in species for which embryonic stem cells are not available.
However, the potential of the technology is only beginning to be explored. Already, there is talk of using the technologies to control malaria by creating “gene drives” (3). These are DNA sequences, which can be introduced into the genome of a mosquito. Using CRIPR-Cas9 technology, such “gene drives” can be constructed to contain all the information necessary to create targeted genomic mutations for malaria resistance. The goal would be to release the resultant transgenic mosquitoes into the wild with the hope that the genetically manipulated mosquitos would mate with wild mosquitos. Because of the engineered sequences in the “gene drive”, all subsequent progeny from the matings of transgenic mosquitoes would be resistant to malaria infection. In an organism like the mosquito, which has a relatively rapid reproductive cycle, it might be possible to make all members of the species resistant to malaria infection within a few years. However, the environmental consequences of such a process have yet to be explored and this needs to be well developed before such a project is allowed to proceed.
Equally challenging is the prospect of using this technology to manipulate animal or human embryos or mature tissue to correct genetic diseases (4). It is theoretically possible for the CRISPR-Cas9 system to correct diseases like sickle cell or cystic fibrosis, which are caused by single base pair mutations. More frightening is the eugenic potential of the technology to engineer almost any polymorphism into the embryo of an organism. The era of “bespoke” genes is now at least within the realm of possibility and it will be a challenge of both science and society in general to apply this technology in an ethical way.
Farmington, CT, USA
A CRISPR Revolution
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