The Use of Programmed Cas9 through crRNA for Genome Editing
The research conducted by Jinek et al (2012) was done with the intention of working on a precision based method for cleavage of DNA at targeted sites. The concept was based on the idea that adaptive defense systems had been created for the purpose of protecting bacteria and archea from viruses and plasmids. This protection is provided by RNA that is small and has been given a specified sequence for detecting and then silencing nucleic acids which are foreign to the system. During the process, cleavage takes place as CRISPR RNA molecules are shortened and then paired with program space or sequences that are complementary and reflect the viral and plasmid targets. The purpose of the study conducted by Jinke et al (2012) was to examine the potential for the use of the endonuclease Cas9 can be used by programming them with one RNA molecule with the intention of cleaving specific DNA sites. The hypothesis of the research is that a simple RNA-based system could be created that would leave the DNA and could be used for targeting and editing the genome.
Jinek et al (2012) write that “Cas9, the hallmark protein of type II systems, has been hypothesized to be involved in both crRNA maturation and crRNA-guided DNA interference” (p. 816). They go on to write that although Cas9 has a relationship with crRNA maturation, it has not been studied its relationship to DNA destruction, nor whether or not it can be used DNA. The researchers examined purified Cas9 protein that was taken from Streptococcus pyogenes to determine if it was possible for cleavage of the plasmid DNA or possibly “an oligonucleotide duplex bearing a protospacer sequence complementary to a mature crRNA, and a bona fide PAM” (Jinek et al, 2012, p. 816). This pivotal experiment showed that the mature crRNA on its own was unable to create “Cas9-catalyzed plasmid DNA cleavage” (Jinek et al, 2012, p. 816). Therefore, it was necessary to look at other approaches for determining whether or not this hypothesis could be supported.
The researchers used extensive controls and experimental frameworks to come to the conclusion that they could support their hypothesis through a dual RNA structure that have the ability to accomplish site-specific double-strand breaks within target DNA. Alternative methodology was proposed by the researchers who stated that RNA programmed Cas9 could potentially be used for genome editing and gene targeting. After five successful cases in which the Cas9 was programmed with chimeric RNAs were able to cleave the plasmid at the targeted site, the next step would be to design an experiment in which practical application of the concepts was studied. While cleavage was studied, whether or not it could end up in editing of the genome was not fully explored. Therefore, additional experiment in which the editing of the genome and an examination of whether or not the edit would have a positive effect would need to be conducted. Therefore, the control would be to examine DNA that was not edited in comparison to the behavior of DNA that had been edited.
The ability to create a system and the value of that ability can be two different outcomes. Success would come from understanding whether or not the attitude genome could then be trusted to behave in a way that was expected once it had been edited. Potential for being able to program RNA to act as a catalyst for ending the manifestations of certain DNA sequences could provide opportunities for making changes related to how certain potentials have an effect on life. Therefore, the next step is to determine whether or not this editing ability is going to have value as a research tool for turning off certain types of potentials that can lead to negative outcomes. Further evaluation will lead to an understanding of the value of this kind of editing as practical tool in a variety of DNA based applications.