Introduction
The treatment for multitudes of diseases and disorders resulting from genetic reasons have been ambiguous since long as the only cure could be the correction of genes and DNA in the specific region. Gene therapy gave a promising horizon to the correction of such genetic defects whereby molecular tools are applied to reverse the mutation or any other changes in the DNA. The advancing biomedical research have offered several tools for gene therapy since last few decades that have been able to perform the desired roles but with one or the other limitations. Lately, CRISPR-Cas9 based tools for gene splicing and performing various genetic editing has paved new path for genome editing that is proving to be above par in specificity, flexibility, simplicity and efficiency along with being economical as compared to other prevalent techniques. In fact, it can be employed for turning the dream of creating designer babies into reality but might take considerable time to come into practicality.
What is exactly CRISPR-Cas9?
Discovery dating back to 1980’s in bacteria Escherichia coli, CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats that are around 20 bps long, with Cas (CRISPR associated proteins) genes being associated with them. They collaboratively impart adaptive immunity in around 40% bacteria and 90% archaebacteria (Zhang, Wen and Guo, 2014 p.41). They gained popularity since 2007 when Barrangou and associates demonstrated their role in developing immunity against bacteriophage in Streptococcus thermophiles. Cas protein families are widespread and more than 40 have been identified so far (Zhang, Wen and Guo, 2014 p.41). The best of Cas genes has been identified in strep throat causing bacteria Streptococcus pyogenes and is popular as Cas9 (Zhang, 2015) or Csn1 that contains HNH and Ruv-C like nuclease domains (Zhang, Wen and Guo, 2014 p.41). CRISPR with Cas9 protein is popular as the modern genetic tool CRISPR/Cas9. There are three major CRISPR mechanisms known but the TypeII has been widely adopted as it employs only Cas9 enzyme for silencing the genes (Reis et al, 2014 p.1) in contrast to other two types I and III that employ huge numbers of complexes (Doudna and Charpentier, 2014 p.3). The system is effectively employed for genome editing functions like DNA insertion, deletion, replacement, labelling, targeting and modulation (Doudna and Charpentier, 2014 p.2).
How CRISPR Functions?
Bacterial immunity via CRISPR
CRISPR are the DNA sequences that are repeated many times with characteristic spacer sequences between the repeats as well (Zhang, 2015). On studying, these protospacer sequences in between the repeats were found to be of bacteriophages. This way the records of virus attacks are stored in the bacterial genome so that when encountered again, the bacteria can defend self through splicing of viral genomes by cas proteins. Cas sequences are embedded nearby CRISPR collection of repeats. In case of a foreign pathogenic attack, the Cas sequences are expressed into Cas proteins that process into Cas enzymes acting as endonucleases. Also, the CRISPR loci genes are transcribed with characteristic spacer sequences and repeats into CRISPR RNA’s (crRNA). Trans-activating crRNA (trRNA) are also required for maturation of crRNA from a common transcript (Reis et al, 2014 p.2). Figure 1 shows brief molecular mechanisms taking place while foreign DNA is destroyed by Cas endonucleases. The Cas enzymes pick these viral origin RNA’s and travels across the cell. As it encounters another viral counterpart matching CRISPR viral origin RNA, then it binds on recognizing PAM (Protospacer Adjacent Motif) sequences and splices a 20 nucleotide long region via double stranded breaks, thus disabling the viral multiplication in the host bacterial cell. The breaks are then repaired through non-homologous end joining pathways.
Figure 1. Demonstration of CRISPR/Cas9 in vivo functioning using crRNA and trRNA essentially (Ries et al, 2014 p.1).
Using CRISPR in genome editing
The crRNA like sequences have been created by researchers Doudna and Charpentier in 2012 (p.3) by combining crRNA and trRNA into single guide RNA i.e. sgRNA or gRNA that comprise a 5’ site sequence for DNA binding and 3’ site RNA duplex for Cas9 recognition (p.1). These guide RNA’s direct Cas9 proteins to any desired genome location.
This RNA based directed genome editing is anyhow simpler than the other comparable protein based techniques as generating target specific RNA is anytime simpler than generating protein motifs, synthesizing them and validating for the same (Doudna and Charpentier, 2014 p.1). Stable mutations are created and transmitted to offspring by plasmids that are prepared expressing Cas9 and crRNA and co-delivered in the target genomes (Zhang, Wen and Guo, 2014 p.42). Transcription regulation is also performed by this system (Zhang, Wen and Guo, 2014 p.43).
Other Genome Editing Techniques
Since the DNA was unveiled, there have been continuous efforts to devise techniques for its modifications and alterations as all the metabolic mechanisms, diseases, disorders and overall survival is dependent on the genes embedded in the DNA. A slight deviation at times like that of single point mutations may also lead to fatal conditions (Hsu, Lander and Zhang, 2014 p.1262). This has tempted researchers to develop site-specific modifications. The initial steps taken towards this goal was wide development of DNA isolation techniques, PCR, electrophoresis, Western & Northern blotting and whole genome sequencing techniques for gene isolation, multiplication and mutation introduction along with studying of the genome functions and structure.
Gradually, genome editing in one or the other became the need of the hour in various fields including clinical, research and development, therapeutics, biotechnological processes, bioprocessing and much more (Doudna and Charpentier, 2014 p.2). The traditional approach was targeting genes for homologous recombination. But, it was a random process and there was a chance occurrence resulting with few mutated cells. Thus, a more specific and efficient approach was required and subsequently small molecules like oligonucleotides and interference techniques using small interfering RNAs came up but RNAi proved to be temporary, incomplete and having unpredictable off-site mutation (Gaj, Gersbach and Barbas, 2013 p.397). Then the more advanced techniques known as the site directed zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) were developed. Above all, CRISPR system has grabbed the sight of every biologist due to its benefits above any other genome editing technique. The most important and common in all the genome editing techniques is the filling of double stranded gaps (DSB) created during the process via either error prone non-homologous end joining (NHEJ) or error free homology dependent repair (HDR) as these repair mechanisms are inherent in all living cells that otherwise would lead to cell death (Doudna and Charpentier, 2014 p.1). Moreover, ZFN and TALEN are nuclease based systems whereas, CRISPR is RNA directed. Table 1 notes the major landmarks in the field of genome editing.
Zinc-finger nucleases (ZFN)
The zinc finger motifs naturally occur in many protein factors that have been exploited in this genome editing method. The sequence specific DNA binding zinc finger domain of transcription factor is fused with non-specific DNA cleaving domain of FokI endonucleases (Gaj, Gersbach and Barbas, 2013 p.398). It targets specific DNA sequences as it can modify ZFN nucleases specific for a triplet base sequence. 64 zinc finger motifs have been gathered each corresponding to a protein codon. DSB’s are created and filled via error prone NHEJ or HDR mechanisms. It requires an elaborated screening for identification of correct DNA-protein binding (Hsu, Lander and Zhang, 2014 p.1263).
Transcription activator-like effector nucleases (TALEN)
Quite the same, TALEN tends to fix FokI endonucleases to TALE proteins. Each TALE protein is specific for a DNA sequence and hence making TALEN also sequence specific, thereby offering the editing tools (Gaj, Gersbach and Barbas, 2013 p.399). TALEs naturally exist in bacterial Xanthomonas species. The protein motifs are around 35 amino acid repeats that bind a base pair. It is advancement over Zinc-finger as it recognizes a single base over triplet by the later. But, it suffers from context dependant specificity and is labour intensive and costly (Hsu, Lander and Zhang, 2014 p.1263).
CRISPR Effectiveness Criterions
Simplicity
The mechanism of CRISPR system is easily understandable and employs only three basic components for performing its action namely Cas9 protein, crRNA and trRNA. It has been further simplified by generating guide RNA’s. Cas9 has been found very useful and several variants like Cas9D10A, dCas9 have been marked for specific purposes (Reis et al, 2014 p.2). The two components processed into a single guide RNA is simplified to target the DNA by just altering gRNA sequences, the only requirement being its association with PAM sequence (Doudna and Charpentier, 2014 p.3). Moreover, CRISPR/Cas9 system just requires an alteration in crRNA for various different targets and functions like regulation, modifications, etc. In contrast, the other techniques demand an entire protein-DNA interaction to be remodelled that is a typically complex protein engineering process. The introduction of CRISPR/Cas system into cells is feasible with nucleofection or with peptides penetrating the cells (Doudna and Charpentier, 2014 p.7).
Precision
CRISPR/Cas9 system is highly focussed and precise as unlike other enzymes, it not just works on the recognition of few DNA sequences within the genome but recognizes the gene broadly (Zhang, 2015). The gRNA binds the DNA on the principle of Watson-Crick base pairing (Hsu, Lander and Zhang, 2014 p.1270). This way, the entire genome is not at risk as small sequence patterns can occur many a times in the genome and enzymes specific for such sequences may disrupt the entire genome. Whereas, the CRISP/Cas9 will identify a specific gene as it can recognize upto 20 bases gene sequences. This makes the system precisely target the genes to be removed through guide RNA or repairing an error in the genome by removing with CRISPR and inserting the normal counterpart. Moreover, CRISPR can be used for revealing genome functions and establishing links between the polymorphisms and related phenotypes due to its precise roles (Hsu, Lander and Zhang, 2014 p.1262).
Efficiency
The experimental timings and complications have been reduced through employing CRISPR system along with saving the huge numbers of animal models like mice, fruit flies under experimentation. The trangenics are created quickly without being dependent on random process of homologous recombination within the cell for generating knockout cell lines. Traditionally, it used to take a lot of toil and mice were bred for three generations to get the desired mutation. Using CRISPR, such mutations are obtained even in several genes via a single attempt by inserting targeted CRISPR/Cas9 system in mice embryo cells where both alleles for a gene are modified (Zhang, 2015).
The targets are achieved by this system much more efficiently than the earlier established genome editing techniques like TALEN or ZFN. The later shows maximum efficiency of 50% but CRISPR is capable of around 70% efficiency in the mouse models so far. TALEN and ZFN works via protein model creation which is far more complicated than creating variations of guide RNA’s which are the key targeting molecules in CRISPR system. The efficiency is tested via T7 endonuclease I detection assays and any unwanted mutations induced by CRISPR have found ways for elimination (Reis et al, 2014 p.4). It aids in screening the entire genomes for identifying the gene functions as huge guide RNA library is generated.
Flexibility
CRISPR is an effective and highly efficient technique for genome editing which is evident by the fact that it has been used for desired genetic modifications in multitudes of cell lines ranging from bacteria, yeast, fruit flies, monkeys, mouse, rats, rabbits, pigs, plants, humans and many more. Single point mutations, translocations and inversions can be easily induced using gRNA with Cas9 (Hsu, Lander and Zhang, 2014 p.1269). The scientists had limitations in exploring more organisms at molecular and genetic levels and the ones feasible were only utilized for research. CRISPR can modulate any genetic makeup and eventually can be used in any living cell, thus opening the arrears of research to unlimited scope. In fact, they can be targeted in multiples with differing sgRNAs associated with Cas-9 making multiplexing possible (Doudna and Charpentier, 2014 p.5).
Economical
In addition to the discussed advantages, CRISPR-Cas modification systems are much cheaper than comparable techniques like ZFN and TALEN. ZFN were once thought to be very efficient but cost a substantial amount of US$5,000 and TALEN kits including TALEN plasmids have also been costing ~$500 (Wang, 2015). In contrast, the RNA guided CRISPR system is available by spending a thrifty amount of $30 that in itself makes it approachable for large and small experimentations by researchers throughout the globe. A breakthrough has been seen in CRISPR acceptability since 2012 leaving behind ZFN and TALEN but still far behind induced pluripotent stem cells technique (Wang, 2015).
A Case Study
The genome engineering of commercially useful yeast Yarrowia lipolytica affirms all the above mentioned aspects as its genome is difficult to edit but CRISPR/Cas-9 made it possible (Schwartz et al, 2015 p.356). It became significant due to markerless tool pCRISPRyl for gene disruption and integration via markerless homologous recombination with an efficiency of >65%. The promoter of SNR52RNA polII was utilized for expressing gRNA on a plasmid as it eases the excision from primary transcripts. Three promoters RPR1, SCR1 and SNR52 were truncated and hybridized with tRNAGly resulting into namely RPR1’, SCR1’ and SNR52’ with tRNAGly that were used to study expression of disruption efficiencies. The disruption of gene PEX10 was examined by insertion of CRISPR/Cas9 system and maximum was observed when SCR1-tRNAGly & SNR52’-tRNAGly promoters were used (Schwartz et al, 2015 p.357). The lack of efficiency was seen on disrupting PAM sequences.
Conclusion
The health care sector has been facing several limitations and long duration for effective treatments. There was an urgent need for a therapy tool that could fasten the process of genetic modification via specific targeting consuming less time compared to the prevailing complex and lengthy techniques. CRISPR has been rightfully welcomed throughout the biological field whereby it has revolutionized the molecular procedures. It is accompanied with all the qualities that was under investigation including simple procedure, effective applications, efficient targeting of genetic modifications and flexibility to be employed in widest range of cell lines. The applications in plants, fungal and algal systems will enhance the agriculture based biotechnological developments along with advantages for pharmacological studies (Doudna and Charpentier, 2014 p.1).
CRISPR system is not a synthetically created tool by researchers but it’s a natural occurring mechanism in bacterial cells. This makes it easily approachable for all the living elements within the ecosystem. Thus, it has the potential to do the genetic modifications and genome editing in almost every living cell. Hence, more research on this system will involve discovering or manipulating the existing Cas classes to develop future variants that are all the more specific and targeted ruling out the minor drawbacks that exits. Drawbacks include the errors that occur at the end when repair mechanisms for filling the DSB come into place. This potential should be evaluated for use in various other applications that make our lives better as the most important medical use for curing genetic diseases will still take some time for approval as direct modification of human DNA is still under ethical debate.
References
Doudna, A, J., and Charpentier, E. The New Frontier of Genome Engineering with CRISPR-Cas9. Science. 2014. 346(6213):1-9.
Gaj, T., Gersbach, A, C., and Barbas, F, C. ZFN, TALEN and CRISPR/Cas-based Methods for Genome Engineering. Trends in Biotechnology. 2013. 31(7):397-405.
Hsu, D, P., Lander, S, E., and Zhang, F. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell. 2014. 157:1262-1278.
Reis, A., Hornblower, B., Robb, B., et al. CRISPR/Cas9 and targeted Genome Editing: A New Era in Molecular Biology. New England Biolabs Inc. 2014. 1:1-5.
Schwartz, M, C., Hussain, S, M., Blenner, M., and Wheeldon, I. Synthetic RNA polIII Promoters Facilitate High Efficiency CRISPR/Cas9 Mediated Genome Editing in Yarrowia lipolytica. ACS Synthetic Biology. 5:356-359.
Wang, B. Disruptive CRISPR Gene Therapy is 150 Times Cheaper than Zinc Fingers and CRISPR is Faster and more Precise. Coverage of Disruptive Science and Technology, Next Big Future. 2015. Accessed at http://nextbigfuture.com/2015/06/disruptive-crispr-gene-therapy-is-150.html on 3 May 2016.
Zhang, F., Wen, Y., and Guo, X. CRISPR/Cas9 for Genome Editing: Progress, Implications and Challenges. Human Molecular Genetics. 2014. 23(1):40-46.
Zhang, S. Everything you need to know about CRISPR, the new Tool that edits DNA. 2015. Gizmodo. Accessed at http://gizmodo.com/everything-you-need-to-know-about-crispr-the-new-tool-1702114381 on 2 May 2016.
