The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system has been rapidly developed, opening new avenues for gene engineering. In recent years, 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). CRISPR-Cas9 technology has evolved to create a simple, RNA-programmable method to precisely mediate genome editing in mammalian cells. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. Even if very efficient, this system is not completely immune to errors, so understanding the possible weak sides could be helpful to prevent all potential off-target effects.
The CRISPR-Cas9 system includes the RNA-guided Cas9 nuclease, which binds to specific DNA sequences (complementary to the RNA-guide sequence) and creates double-stranded breaks (DSB) on the DNA. The dsDNA breaks can be repaired via homology-directed repair (HDR) or nonhomologous end-joining (NHEJ). Based on this principle, the Cas9 and the guide-RNA were modified in various ways to improve the efficiency and specificity of this system, to expand its potential for different applications. This system can be used for altering specific genetic loci through insertions, deletions, point mutations, and sequence inversions. More recently, the system was modified to act as a genome regulator, by tethering effector domains to the Cas9 or guide-RNA, and as a visualization tool by fusing with marker molecules. This multiplex capacity of engineering CRISPR-Cas9 enabled scientists to apply this system for genome modifications in a variety of organisms.
The CRISPR-Cas9 system comprises a robust technology that has been used in diverse and innovative applications in biology. It has incomparable advantages over other gene editing tools. For example, the CRISPR-Cas9 system has more target sites than ZFNs and TALENs, and Cas9 has many variants that can be used in a variety of studies. Moreover, the system is extremely easy to use and can be executed in almost any laboratory. Cas9-based tools have greatly enhanced our ability to perform systematic analyses of gene function, as well as to reproduce tumor-associated chromosomal translocations precisely. This technology has also paved the way for the dissection of redundant gene functions, epigenetics and possible gene therapy. The CRISPR-Cas9 technology is currently the simplest, most precise, and versatile method of genome editing in a variety of cells and organisms, both in vitro and in vivo.
To date, the CRISPR-Cas9 system has already shown itself to comprise a robust and ﬂexible tool for genome editing and gene regulation. With further research on CRISPR, however, it became apparent that this technology was not as easy as once assumed. Despite the many advantages of this system, there are some challenges to the current Cas9-based tools. A large number of studies have investigated diverse aspects that affect the efﬁciency and speciﬁcity of CRISPR-Cas9 system, such as Cas9 activity, target site selection and sgRNA design, delivery methods, off-target effects, and the incidence of homology-directed repair (HDR). Many of the breakthroughs in the genome engineering sphere have been viewed as huge scientific triumphs, but the translation of these technologies from the bench to the bedside is not without ethical, legal and social issues requiring vigorous debate.
One potential limitation of CRISPR-Cas9 technology is that the approach may create off-target effects. These off-target effects might play a role in recognizing and destroying hypervariable viral nucleic acids or plasmid DNA, which is beneﬁcial to bacteria and archaea. However, for biological studies and genetic therapies, off-target phenomena generate undesired mutations at random sites, thus impacting precise gene modiﬁcation. We must be wary of the potential consequences of off-target effects, lack of specificity in targeting, incomplete targeting, and so on, all of which could have devastating effects on patients. Scientists will be looking for improved nucleases to increase safety and efficacy. In particular, the improved nucleases hold promise for reducing off-target effects, which would improve the effectiveness of the target gene edit, and reduce unintended consequences. Greater accuracy will also improve our ability to characterize mechanism and to monitor efficacy and safety, including over multiple generations.
Ethical concerns about germline gene editing is also a contentious issue. There are myriad concerns about the introduction of heritable modifications should any of the manipulated embryos be used in reproduction. There are concerns about the risk of errors and (unintended) consequences, not only for the resulting child(ren) but also for humankind. In the latter context there are concerns about the risk of exacerbating problems of racism, sexism, health inequality, and so on, as a direct consequence of who will and who will not have access to the technology. A bigger obstacle to the emergence of "designer babies" and Gattaca-type dystopian futures: the principles of evolution. Gene editing could ruin human evolution. Though some challenges remain ahead, the application of this technology to several aspects of sarcoma biology, ranging from basic research to clinical and translational applications, offers numerous exciting opportunities for a better understanding and potential treatment of these devastating diseases.
1. SK Vasiliou et al. CRISPR-Cas9 System:Opportunities and Concerns. Clinical Chemistry. August 22, 2016;62:10.
2. S A Nicholson and M S Pepper. CRISPR-Cas: Revolutionising genome engineering. SAMJ. September 2016, Vol. 106, No. 9:870-871.
3. R. Peng et al. Potential pitfalls of CRISPR-Cas9-mediated genome editing. FEBS Journal. November 4, 2015;283(7):1218-31.
4. Liu et al. Development and Potential Applications of CRISPR-Cas9 Genome Editing Technology in Sarcoma. Cancer Lett. April 2016; 373(1):109-118.