The discovery of Cas9, a large protein that involves in all processes the type II system of Clustered Regularly Interspaced Short Palindromic Repeat, an adaptive immune system from bacteria and archaea, is a paradigm shifting molecular tool in the world of cell biology research due to its potential for various biotechnology applications. As an RNA-guided DNA targeting endonuclease, Cas9 can essentially cleave any DNA sequence of interest based on Watson-Crick base pairs using the guide region of a single guide RNA (sgRNA), which is comprised of the processed sequence of CRISPR RNA (crRNA) that carries the spacer sequence that requires searching for the targeting DNA, covalently linked to a trans-activating crRNA (tracrRNA) that provides a scaffold for Cas9 protein to bind to the crRNA. While Cas9 has tremendous application in any DNA-associated problems, which includes eradicating genetic diseases or gene mutations, Cas9 has an inherent off-target DNA cleavage – that is cleaving a potential DNA target that may have a similar but incorrect sequence compared to the on-target DNA sequence – due to one or multiple mismatches between the gRNA and the targeting DNA. As a result, it poses a concern regarding its use in gene therapy in human or other animal systems. We chose to address this DNA target specificity and efficiency issue by establishing and studying through a novel Cas9 system from a subtype different than the established systems – a type II-C Cas9 from thermophile Acidothermus Cellulolyticus 11B (AceCas9). Further information of the classification of Cas9, molecular processes that involved Cas9, the significance of Cas9 and this research project are addressed in Chapter 1. Without any previous studies on AceCas9, we established the in vitro biochemical functions of AceCas9 through combinatorial methods to determine 1) the sgRNA sequence that yields a Cas9 RNP, 2) a functional PAM sequence that permits AceCas9 to cleave dsDNA through an in vitro DNA library assay, 3) determine the target specificity of PAM and target DNA in vitro through mutational analysis on oligo DNA substrates, and 4) environmental conditions that influence target efficiency of AceCas9 in chapter 2. The results showed that AceCas9 recognized a novel, cytosine-specific PAM sequence (5’–NNNCC–3’) and proves to be functional in vitro. AceCas9 depends on limited divalent cations for DNA cleavage, yet it proves to be functional at a wider range of temperature, from 37 °C to 60 °C. To further establish DNA cleavage specificity and efficiency, we performed cleavage assays as well as a series of single-turnover kinetics assays to determine how specific and efficient AceCas9 cleaves plasmid DNA substrates with various mutation and/or DNA topologies under in vitro condition in chapter 3. Results showed that substrates with higher helicity permit AceCas9 to cleave those substrates quicker, yet decrease AceCas9 target specificity. Finally, with the hypothesis that the sgRNA may influence target efficiency and specificity, we performed both in vitro and a bacterial-based in vivo assays to determine how elongation on the guide length may influence DNA interference by AceCas9. We demonstrated that an elongated guide length from 20-nt to 24-nt significantly improves AceCas9 DNA targeting efficiency both in vitro and in vivo, but it does not contribute significantly in target specificity. Intriguingly, AceCas9 can only be functional in vivo with either a 24-nt or 26-nt guide in sgRNA, suggesting that AceCas9 is selective to the length of its spacer, contrary to its counterparts.
