Mutagenesis and Functional Analysis of a CRISPR Endonuclease in Yeast

Genome editing offers the possibility of effectively combating disease. Current approaches require the design and expression of novel proteins for each genomic site to be edited, limiting their efficiency and applicability. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/cas genetic interference pathway found in archaea and bacteria may enable a powerful genome editing approach that is not only specific, but is also directed by simple base pairing of a small RNA. Cas9 belongs to a Type II CRISPR/Cas system, and has been identified as the sole cas gene needed for RNA-directed DNA cleavage in bacteria. We hypothesized that the wild-type Cas9, without any mutations, will be expressed and will successfully cleave DNA in eukaryotes such as budding yeast (Saccharomyces cerevisiae). However, in the presence of certain Cas9 mutations, we expect no Cas9 function and therefore no DNA targeting. We are using the Cas9 protein from Streptococcus pyogenes (SF370) for our experiments in eukaryotes. Following site-directed mutagenesis of Cas9 and transformation into yeast, Cas9 will be labeled with an HA tag, and we will monitor its expression and its ability to cleave specific genomic loci in vivo when programmed with a small RNA. We expect to see a loss of Cas9 function, and therefore a loss of RNA-directed DNA double-strand break induction. By understanding the important components of the CRISPR/Cas system, we propose a potent genome editing mechanism for eukaryotic cells to further enhance our ability to investigate gene function and to fight disease.