IntroductionThe CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages, and provides a form of acquired immunity. CRISPR spacers recognize and cut these exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea.
Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins are found in many bacteria and most archaea. The CRISPR-Cas systems use sequences derived from plasmids and phages to activate Cas endonucleases to neutralize those plasmids and phages via RNA-guided sequence-specific DNA cleavage, thus blocking their transmission and creating a simple acquired immunity. The CRISPR/Cas system has been used for gene editing (adding, disrupting or changing the sequence of specific genes) and gene regulation in species throughout the tree of life.By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be relatively cheaply cut at any desired location.CRISPR has a number of potential applications including treating genetic diseases, fighting infections, and increasing food crop yields, but the application of this method is accompanied by ethical concerns.
The CRISPR/Cas9 system for targeted genome editing
Although recently-developed programmable editing tools, such as zinc finger nucleases and transcription activator-like effectors nucleases, have significantly improved the capacity for precise genome modification, these techniques have limitations. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technology represents a significant improvement over these other next-generation genome editing tools, reaching a new level of targeting, efficiency, and ease of use. The CRISPR/Cas9 system allows for site-specific genomic targeting in virtually any organism.
The type II CRISPR/Cas system is a prokaryotic adaptive immune response system that uses non-coding RNAs to guide the Cas9 nuclease to induce site-specific DNA cleavage. This DNA damage is repaired by cellular DNA repair mechanisms, either via the non-homologous end joining DNA repair pathway (NHEJ) or the homology directed repair (HDR) pathway.
The CRISPR/Cas9 system has been harnessed to create a simple, RNA-programmable method to mediate genome editing in mammalian cells, and can be used to generate gene knockouts (via insertion/deletion) or knockins (via HDR). To create gene disruptions (Figure 1), a single guide RNA (sgRNA) is generated to direct the Cas9 nuclease to a specific genomic location. Cas9-induced double strand breaks are repaired via the NHEJ DNA repair pathway. The repair is error prone, and thus insertions and deletions (INDELs) may be introduced that can disrupt gene function.
Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities.Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterogonous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease. Role of CRISPR in Gene therapy
Gene therapy involves manipulating DNA or RNA for human disease treatment or prevention. The strategies of gene therapy are diverse, such as rectifying, replacing or deleting the culprit genes in genetic diseases, producing disabling mutations in pathogen genomes to combat infectious diseases or inducing therapeutic or protective somatic mutations. It is a promising therapy for a wide range of human diseases including hematological diseases, cancer, AIDS, diabetes, heart failure, and neurodegenerative diseases.
Correcting monogenic disorders
Monogenetic disorder is caused by single gene defects. Compared with polygenic diseases such as cancer, monogenic disorders are more amenable to gene therapies. Currently, the correction of monogenic disorders represents the most translatable field in CRISPR-Cas9-mediated gene therapy.
Somatic gene correction in adult animalsCRISPR-Cas9 technology can also mediate somatic gene corrections in adult animals, bypassing embryo manipulations. [Yin ,] for example, delivered CRISPR-Cas9 agents and a homologous donor template (in order to increase HDR rate) into adult mice with hereditary tyrosinemia via tail-vein hydrodynamic injection, resulting in gene corrections in 0.25% of liver cells initially, and in 33.5% of liver cells 33 days post injection (possibly due to selective advantages imposed by the correction), which was sufficient to rescue the disease phenotype. This method is more translatable to human therapeutics because it does not involve embryo manipulations.
The technology has been used to functionally inactivate genes in human cell lines and cells, to study Candida albicans, to modify yeasts used to make biofuels and to genetically modify crop strains
CRISPRs can add and delete base pairs at specifically targeted DNA loci and have been used to cut as many as five genes at once. CRISPR's low cost compared to alternatives is widely seen as revolutionary.
Selective engineered redirection of the CRISPR/Cas system was first demonstrated in 2012 in the following applications:
· Immunization of industrially important bacteria, including some used in food production and large-scale fermentation
· Cellular or organism RNA-guided genome engineering. Proof of concept studies demonstrated examples both in vitro and in vivo.
· Bacterial strain discrimination by comparison of spacer sequences
"CRISPRi" like RNAi, turns off genes in a reversible fashion by targeting but not cutting a site. RNA-guided CRISPR associated nuclease Cas9 is an effective way of targeting and silencing specific genes at the DNA level.In bacteria, the presence of Cas9 alone is enough to block transcription, but for mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA, called promoters that immediately precede the gene target.
Cas9 was used to carry synthetic transcription factors (protein fragments that turn on genes) that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different spots on the gene's promoter.