CRISPR Revolution

What makes CRISPR so revolutionary is that it's incredibly precise: The Cas9 enzyme mostly goes wherever you tell it to. And it's incredibly cheap and easy: In the past, it might have cost thousands of dollars and weeks or months of fiddling to alter a gene. Now it might cost just $75 and only take a few hours. With its unprecedented efficiency and stunning ease of use, DNA/RNA editing technology based on the prokaryotic CRISPR-Cas system is completely revolutionizing genome engineering, especially CRISPR-Cas9 system. In little more than a year, CRISPR-Cas9 editing has been implemented in a multitude of model organisms and cell types and has already started to supplant incumbent genome editing technologies, such as TALENs (transcription activator-like effector nucleases) and ZFNs (zinc finger nucleases).

The CRISPR-Cas technology is derived from a bacterial pathway that allows the organism to detect and degrade invading genetic material. Initial work by Jinek et al. has seen the progression of CRISPR-Cas from an interesting bacterial phenomenon to a potentially useful molecular tool. Later work by Cong et al. ushered CRISPR-Cas to the forefront of the gene-editing technology race. The CRISPR revolution will change life by bringing together experts from industry, academia and government to discuss the current state-of-the art in the use of the CRISPR-Cas9 system to understand biological pathways, to create cellular and animal models of disease, to develop improved agricultural crops and farm animals and to create new human therapies to correct somatic gene defects that lead to disease. CRISPR-Cas have emerged as a novel class of sequence-specific endonucleases with unparalleled flexibility, cost-effectiveness and ease of application.

Background and Development of CRISPR Revolution

Despite capturing the interest of microbiologists and molecular biologists, the CRISPR-Cas system stayed at least initially under the radar of the general biomedical research community. This suddenly changed when several groups demonstrated the Cas9 protein from Streptococcus Pyogenous could be reprogrammed with synthetic RNAs to generate site-specific double-strand breaks (DSBs) in vitro and in mammalian cells. The CRISPR-Cas9 is an example of a type II-A CRISPR-Cas system and was chosen because a single protein (Cas9) is sufficient to induce cleavage at the target site. In S. Pyogenous Cas9 is complexed with two RNAs, the crRNA, whose specific sequence confers target-specificity, and a 77-nucleotide trans-activatingRNA (tracrRNA), which is required for target cleavage. Fortunately, a single guide RNA (sgRNA) can effectively replace the native RNA duplex, thus providing a versatile binary system to introduce specific DSBs in mammalian cells.

The ability to introduce specific DSBs into the genome of mammalian cells is not entirely new: TALENs and ZFNs engineered to bind and cleave at specific sites have been used previously with success. However, the intrinsic simplicity of the CRISPR-Cas9 system-for which generating a new guide RNA is all that is needed to cleave at a different site-has made somatic genome editing available to virtually any laboratory equipped for basic molecular biology. The only requirement for CRISPR-Cas9 to function is the presence, on the target DNA, of a short sequence known as PAM that has to be located immediately downstream of the sequence recognized by the sgRNA. The PAMs recognized by the S. Pyogenous Cas9 are "NGG" and-with much reduced efficiency-"NAG". This requirement somewhat limits the sequence space that can be edited by CRISPR-Cas9. To overcome this limitation, modified versions of the Cas9 protein have been engineered that recognize different PAM sequences.

Identification of CRISPR-Cas system to change the genome started as whisper, but during the past few years it has grown to a deafening roar and at last there seems to be a technology with genuine potential to revolutionise the field of genome engineering. CRISPR-Cas9 system, as a revolutionary genome editing tool in mammalian tissues, has revolutionized how gene perturbation experiments are conducted at a genome-wide scale and has enabled the unprecedented creation of genetically-altered non-human primates for pre-clinical studies. The CRISPR-Cas9 system is revolutionizing genomic engineering and equipping scientists with the ability to precisely modify the DNA of essentially any organism. This gene editing could potentially confer genetic advantages that previously took large amounts of evolutionary time (and perhaps a bit of luck), taxing genetic breeding strategies, or bulkier and more complex genomic editing tools to acquire.

Recently, with the discovery of the nuclease Cas13a (C2c2), scientists have obtained a system that enables sequence-specific cleavage of single-stranded RNA molecules. Cas13 is a newly identified CRISPR effector that specifically cleaves single-stranded RNA in eukaryotic cells. CRISPR-Cas13 is a robust, precise and versatile RNA-targeting system, opening up a wide range of new possibilities. Compared with previous technologies for RNA manipulation, CRISPR-Cas13 offers several advantages. Its modular composition consisting of a single protein effector module and an RNA guide module enables not only a simple and fast design, but also large scalability by the generation of whole libraries of different guide RNAs. Compared with RNAi, CRISPR-Cas13 mediated manipulations are not restricted to targeting cytoplasmic transcripts, but non-coding nuclear transcripts and pre-mRNA can also be targeted by simply adding a nuclear localization signal.

Problems in CRISPR Revolution

As with any new technology, the initial phase of excitement is invariably followed by a more careful evaluation of its limitations. In the case of CRISPR, reducing or at least taking into account off-target effects is clearly of great importance. In addition, the translation of these technologies from the bench to the bedside is followed by ethical, legal and social issues. Firstly, can we predict the ultimate consequences of gene editing on the evolution of the human race? Secondly, should we allow embryonic/germline engineering, or only permit somatic cell engineering? The genome engineering community has reached a consensus that is genome engineering for research purposes should be allowed in both somatic and embryonic cells, but with important ethical concerns and the concerns around safety, genome engineering for therapeutic applications should be restricted to somatic cells and so on.

The Future of CRISPR Revolution

Major factors underlying the CRISPR-Cas9 genome editing revolution include the compactness, simplicity, and targeting flexibility afforded by this system. The implications for enhanced Cas9 targeting specificity and binding efficiency are exciting and establish a basis for the further optimization of the Cas9-sgRNA system and the development of next-generation CRISPR tools. Future studies will determine the potential of various natural or engineered Cas9 proteins, sgRNA molecules, and their respective PAM sequences for increased sequence recognition specificity and DNA binding efficiency, and possibly the generation of short Cas9 homologs for convenient packaging and delivery. Notwithstanding the CRISPR craze of 2013, this dynamic field is off to an effervescent start for 2014, and these latest findings open new engineering avenues for CRISPR-Cas9 and set the stage for further applications in synthetic biology, translational research, and next-generation genome engineering.

An important benefit of the CRISPR-Cas system is that it provides an experimental pipeline to rapidly model and functionally test newly identified mutations in oncogenes and tumor suppressor genes. This could have important implication in personalized medicine where it could be used to guide the choice of the optimal targeted therapy for individual cancer patients. Despite a number of important ethical and practical concerns, CRISPR technology remains potential to shape the future of molecular medicine. This has obvious influence on the ability to study gene function: genes can be knocked out, specifically altered for gain of function, modified for conditional control or tagged for tracking of the protein product. This has also had an immediate and significant impact on reverse genetics. These technologies will be greatly valuable in decoupling the complexity of eukaryotic gene regulation and for applications such as reprogramming gene networks to direct changes in cell phenotype.

CRISPR Revolution Related References

1. Ram Kannan and Andrea Ventura. The CRISPR revolution and its impact on cancer research. Swiss Med Wkly. 10 December 2015;145:w14230.
2. Munshi. CRISPR/Cas9: A Revolutionary Disease-Modifying Technology. Circulation. 2016 September 13; 134(11): 777–779.
3. Rodolphe Barrangou. Cas9 Targeting and the CRISPR Revolution. Science. 16 MAY 2014;344, 707-708.
4. S A Nicholson and M S Pepper. CRISPR-Cas: Revolutionising genome engineering. SAMJ. September 2016, Vol. 106, No. 9:870-871.
5. Felix Wolter and Holger Puchta. The CRISPR/Cas revolution reaches the RNA world: Cas13, a new Swiss Army knife for plant biologists. The Plant Journal (2018) 94, 767–775.