The main purpose for this research paper is to find out the role of CRISPR: RNA programmable genome system with two RNA chimeras (tracrRNA and crRNA) in an associated protein called Cas9 endonucleases that provide the microorganisms like bacteria and archaea with adaptive immunity defense to protect them against any foreign attacker like viruses or plasmids.
The hypothesis they tested in this paper was done to study the mechanism of dual RNAs to cleavage a specific site in DNA and Oligonucleotides that has a complementary protospacer sequence to the maturation crRNA and the functional PAM (protospacer-associated motif). Furthermore, the test result shows that crRNA is dependent on tracrRNA (that direct the Cas9 protein during the cleavage processing) so tracrRNA is required to complete the pairing and complementary sequence of crRNA. The supposedly pivotal experiment can be in figure 2B, there are two Cas9 domains: HNH cleaves the complementary DNA strand and the other RuvC which cleavage the noncomplementary DNA strand, specific base pairing positions 5 to 12 are important for efficient Cas9 binding and target recognition according to the shorter RNA chimeric limitation. Although, they designed five chimeric RNAs to target the fluorescent protein to cleavage the GFP gene with a plasmid.
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Otherwise, most of the important points regarding the experiments, the products, and the consequences are covered in each spot, but there are some lacking points in the all five cases of Cas9 in general because I couldn’t understand the main mechanism of dual-RNA programmed between these five cases. I would like to study further with the experimentation by using different features of Cas9 endonucleases that have a combined chimeric RNAs by joining trRNA and crRNA into a single synthetic single guide RNA (sgRNA) that has the target gene and developed chemically modified sequences that can provide additional sgRNA, then doing in vivo transcription on the model organism like Staphylococcus aureus then PCR to produce a purified sgRNA for testing or cell transduction. Also, we can add Zinc-finger nucleases and transcription-activator to improve the cell-mediated immunity.