The ability to engineer genomic DNA in cells and organisms easily and precisely will have major implications for basic biology research, gene therapy, biotechnology and the future of medicine. Technologies for making and manipulating DNA have enabled many of the advances in biology over the past 60 years1. The introduction of genomic sequencing technologies and the generation of whole-genome sequencing data for large numbers and types of organisms has been one of the most important advances of the past two decades regarding genome science. The CRISPR-Cas9 system has proven difficult to use in the lab however, the implications of the technology may change the face of genome science and genetic diseases as we know it.
Since the discovery of the DNA structure by Watson and Crick in 1953, researchers have been searching for ways to make site-specific changes to genomes. The RNA-guided enzyme Cas9, which originates from the CRISPR-Cas adaptive bacterial immune system, is transforming biology by providing a genome engineering tool based on the principles of Watson-Crick base pairing1. The application of CRISPR technology in genome-wide association studies will enable large-scale screening for drug targets, SNPs and potential SNP repair as well as other phenotypes and will facilitate the generation of engineered animal models that will benefit pharmacological studies and the understanding of human diseases among many other things1.
CRISPRs or clustered regularly interspaced palindromic repeats were first described in 1987 by Japanese researchers as a series of short direct repeats interspaced with short sequences in the genome of Escherichia coli2. It wasn’t until 2002 that CRISPRs were found to be numerous in bacteria and archaea at which point they were predicted to play a role in DNA repair3. Upon more investigation into CRISPR loci it was found that they are transcribed and that Cas – which are CRISPR-associated genes – encode proteins with putative nuclease and helicase domains4.
It was soon after proposed that CRISPR-Cas is an adaptive defense system against invading phages and plasmids which functions analogously to the eukaryotic RNA interference (RNAi) systems5. In 2008, mature CRISPR RNAs (crRNAs) were shown to serve as guides which complex with Cas proteins to interfere with virus proliferation in E. coli6. Concurrently in 2008, CRISPR-Cas was harnessed for its DNA targeting activity in Staphylococcus epidermidis7.
Functional CRISPR-Cas loci are comprised of a CRISPR array of identical repeats intercalated with invader DNA-targeting spacers that encode the crRNA components and an operon of Cas genes encoding the Cas protein components8. The protospacer adjacent motif (PAM), a short sequence motif adjacent to the crRNA-targeted sequence on the invading DNA, plays an essential role in the stages of adaptation and interference in CRISPR systems8. In naturally occurring environments, viruses can be matched to their bacterial or archaeal hosts by investigating CRISPR spacers. Experiments in this area have shown that viruses are constantly evolving to avoid CRISPR-mediated attentuation8.
In 2012, the S. pyogenes CRISPR-Cas9 protein was shown to be a dual-RNA–guided DNA endonuclease that uses the tracrRNA:crRNA duplex to direct DNA cleavage9. Trans-activating
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