Investigation of a Type II-C CRISPR-Cas9 on Its Sensitivity to the Epigenetic State of DNA Substrates

CRISPR Cas9 is a bacterial immune system which has been found to be extremely useful for the purposes of genomic editing and DNA detection methods. While it is possible to "program" Cas9 proteins for specific target sites, any site must also contain a match for the Cas9 protein's PAM sequence otherwise the protein will not bind. Preliminary experiments reveal AceCas9, one variant of CRISPR Cas9 isolated from Acidothermus cellulolyticus bacteria with a PAM sequence of 5'-NNNCC-3', is sensitive to the presence of methylation on the fourth-position cytosine in its PAM sequence meaning it will not successfully cleave any target site possessing 5-methylcytosine at this position. DNA methylation is an important epigenetic change which can influence gene expression but current methods of detecting DNA methylation either require harsh reactions involving sodium bisulfite that can degrade sample DNA or methylation-sensitive restriction enzymes which frequently have highly inflexible and non-programmable target sites. This information was used in the creation of a methylation-detection assay which uses AceCas9 protein to "filter" non-methylated DNA by cleaving it, the methylated DNA remaining may then be amplified via PCR, and then analyzed after gel electrophoresis. The detection limits of this methylation assay have been found to consistently detect methylated DNA despite only constituting 1*10⁻⁵% of a given sample. This would allow for the detection of methylation for a single nucleotide to a concentration as low as 1*10⁻⁵% without requiring the harsh reaction conditions involving sodium bisulfite or an inflexible target site required by a methylation-sensitive restriction enzyme.