The CRISPR-Cas system is a form of genetic adaptive defence mechanism, found in bacteria and archaea, against invading nucleic acids and predatory viruses. It allows the host to remember, recognize and fight invading infections. CRISPR, stands for clustered, regularly interspaced, short palindromic repeats, are specific genetic loci found in the genome of most of the species of Prokaryotes and Archaea, as a part of the CRISPR-Cas system (Figure 1). The CRISPR loci consist of short non-contiguous direct repeats (30-40 bp long) interspaced with equally short variable sequences called ‘spacers’ (Horvath & Barrangou, 2010). These spacer sequences are homologous with short genetic sequences of plasmid and viral origins, thereby significantly contributing to the host resistance against the invading nucleic acids. Furthermore, the Cas (CRISPR associated system) genes encode for a large and diverse group of proteins, know to interact with nucleic acids, like nucleases, helicases, polymerases, polynucleotide binding proteins etc. and has RNA-directed activity (Cas proteins possess RNA binding domain) (Rath, Amlinger, Rath, & Lundgren, 2015). Based on the type of Cas protein involved, the CRISPR-Cas system are categorized into three types, I, II and III of which, the Type II, CRISPR-Cas9 system, a DNA targeting system has been found to be the most optimal system biotechnological applications (Rath et al., 2015).
Mode of action of CRISPR-Cas system
The CRISPR-Cas system essentially provide adaptive immunity to the host by a unique self and non-self-recognition system, similar to the eukaryotic RNAi mechanism, and consist of CRISPR loci genetically linked with Cas genes. Overall the CRSIPR-Cas system provides immunity to the host through a three stage process:
- Expression and
- Interference (Marraffini & Sontheimer, 2010) (Figure 2).
In the adaptation stage, a Cas complex cleaves and integrates novel spacer fragments of the invading nucleic acid (plasmid or viral genome) genome within the CRISPR locus at one end. Following subsequent infection of same nuclei acid, the second stage, expression, is activated wherein the CRISPR loci is transcribed as precursor CRISPR-RNAs or crRNAs, which are processed into smaller mature crRNA by Cas proteins. In the last stage of interference, the crRNAs along with another segment tracrRNA bind to Cas9 nuclease protein, forming a crRNA-Cas9 complex and are guided towards the target foreign nucleic acid (plasmid or viral genome) for its degradation. Cas9 brings about degradation in the foreign DNA by binding to it through complementary regions of crRNA and introducing double stranded breaks in it (Charpentier & Marraffini, 2014).
In the CRISPR-Cas9 system, the Cas loci codes for three major proteins, Cas1, Cas2, Cas9 and sometimes a fourth protein, Csn2 or Cas4 (Rath et al., 2015). The Cas9 protein, the signature protein of this system, has both target region recognition and nuclease activity, which allows it to successfully degrade foreign nucleic acids once activated. The recognition of nucleic acid as self or non-self by the CRISPR-Cas system is driven by a small motif next to the target spacer sequence, called CRISPR motifs or protospacer adjacent motif (PAM) whose presence also determines the ability of the foreign nucleic acid to be degraded by Cas9 (Marraffini & Sontheimer, 2010; Rath et al., 2015). The CRISPR-Cas system has been found in approximately 90% of archaeal species and also in around 40% of bacterial species (Horvath & Barrangou, 2010).
Practical applications of CRISPR-Cas technology
Considering the specific intrinsic properties of CRISPR-Cas immunity systems, the first and foremost application is the use of the hypervariability of CRISPR loci for typing isogenic strains. Sofar, CRISPR typing has been done for important bacterial species like Mycobacterium tuberculosis, Yersinia pestis, Salmonella spps. And Corynebacterium diphteriae (Rath et al., 2015). Furthermore, the ability of CRISPR-Cas system to provide immunity to the hosts against phages and invasive genetic elements has been used by the Diary industry for production of important phage-resistant strains of lactic acid bacteria like Lactobacilli spps. (Barrangou & Horvath, 2012).
However, the most prominent application of CRISPR-Cas system is the ability of the unique system to perform sight-specific genetic modification in cells and whole organisms. Common genetic engineering and gene editing tools have played an important role in the field of biology, ranging from applications in biotechnology, medicine to drug discovery. The ability to introduce, delete or perform site specific genomic sequence changes in cells has typically been performed based on sight-specific DNA recognition by oligonucleotides or self-splicing introns or the more recent application of DNA-binding proteins like Zinc Finger Nucleases (ZFN) and TAL effector nucleases (TALENs) (Doudna & Charpentier, 2014). The CRISPR-Cas system, on the other hand, bypasses the problems of protein design, synthesis and validation faced by the engineered DNA-binding proteins and provides an easy to use, faster and cheaper mode of gene editing (Doudna & Charpentier, 2014).
In case of CRISPR-Cas system as a gene modification technology, the crRNA:tracrRNA complex was designed into a single-guided RNA (sgRNA) which consists of the targeting sequence at its 5’ end to be paired with the target DNA, while the double stranded crRNA:tracrRNA at 3‘ end binds to Cas9 endonuclease. As a result, the Cas9 enzyme can be programmed with sgRNA sequence to target any DNA sequence as long as there was a PAM sequence adjacent to it (Doudna & Charpentier, 2014). Owing to this simplicity of mechanism and capacity for multiplexed target recognition, CRISPR-Cas system since its discovery has been proved to work in a variety of systems, ranging from bacteria to human cells (Charpentier & Marraffini, 2014).
CRISPR-Cas gene modification systems have been used for successful targeted mutagenesis and genome modification in bacteria, yeast, plants, animals and human cells (Figure 3). Through gene modification, basic functioning and regulations of genes can be studied, besides elucidating the networks of important metabolic pathways (Jakočinas et al., 2015). Genetic modification using CRISPR-Cas can also help design genetically modified crops like environmental and disease resistant plants as well as for metabolic engineering or metabolic farming (plants or plant cells used as factories for manufacturing of specific proteins or metabolites) (Bortesi & Fischer, 2014). Metabolic engineering can be useful for not only production of important biometarials, but also for economical drug manufacturing in bacterial or yeast factories (Hsu, Lander, & Zhang, 2014). Furthermore, the technology can also be used to successfully design cellular and animal models for research into different causal roles of genetic variations (Hsu et al., 2014). In the field of drug discovery, the multiple locations targeting ability of CRISPR-Cas could help study the additive effects underlying complex polygenic disorders, thereby revealing new drug targets for treatment (Hsu et al., 2014). The most important application of CRISPR-Cas system is its use in gene therapy for treating genetic disorders, by correcting harmful mutations in both embryonic and somatic cells (Harrison, Jenkins, O’Connor-Giles, & Wildonger, 2014). The gene therapy through CRISPR-Cas also offers opportunities for treatment of cancer (deactivation of oncogenes or induction of oncosuppressor expression), induction of protective/ therapeutic mutations in host tissues and engineering certain pathogen genome like HIV for therapeutic purpose (Xiao-Jie, Hui-Ying, Zun-Ping, Jin-Lian, & Li-Juan, 2015).
Future prospects and ethical considerations
Considering the advantages and possible applications of CRISPR-Cas system in gene regulation studies and targeted gene modification, the future prospects for this technology is indeed bright, proven by the large number of researches and applications being published in the past two years. However, the debate over the ethics of gene editing which focuses on the long term ethical and practical consequences of Genetically Modified Organisms (GMOs) has once again renewed. Researchers across the world are more frequently employing CRISPR-Cas technology with ease; to not only study the effects of genes but also to design genetically modified organisms suiting a particular purpose besides developing therapies for important diseases in human. However, the CRISPR-Cas technology is still at its infancy and has a long way to go before it can be applied for large scale practical therapies. This technology faces a number of challenges as an application like delivery to right part of the body, range of impact of the technology on the host genome and the consequences of ‘off-target’ effects of the technology on the human body. All of which have to be addressed before CRISPR-Cas based therapies can be developed and enter clinical trial phases (CNN, 2015).
There has been a wide-spread concern regarding the ethics and consequences of using the gene editing tools in humans. This debate took a turn for worse recently when a group of Chinese researchers declared the mixed results of their attempt of curing thalassemia in human embryos, casting a light on the underdeveloped CRISPR-Cas technology suitable for treating human conditions on a commercial scale. Ethicists argue that application of CRISPR-Cas in human therapy is one step away from developing designer babies. There needs be a regulatory framework for human gene editing technologies that encompasses the technical and ethical requirements inherent to these therapies and also addresses undefined safety mechanisms, augmented risks of multigenerational side-effects and ethical hurdles of designed human embryos (Tomislav, 2016).
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