Using Gene Editing to Create SNPs and Point Mutations in Human Genes Contained in Yeast Artificial Chromosomes

Anja J. van Brabant1, J. Kendall Williams1, Hetal Parekh-Olmedo2, Hannah N. Minch1, Brian C. Matlock1, Eric B. Kmiec1,2 1NaPro BioTherapeutics Inc., Genomics Division, and 2Dept. of Biological Sciences, Delaware Biotechnology Institute, University of Delaware Presenter - J. Michael Hurley, PhD A single nucleotide polymorphism (SNP) in a human gene can alter the sequence and properties of the corresponding protein. These polymorphisms may affect an individual's response to drug therapy or be disease-causing mutations. Using gene editing technology, we can rapidly and efficiently introduce a specific SNP into virtually any gene at any desired position, through the use of proprietary modified single-stranded oligonucleotides. Our gene editing protocol uses a novel dual-targeting strategy to alter genes contained in yeast artificial chromosomes (YACs), which have the largest insert capacity of current cloning systems. As such, introns and regulatory regions, as well as coding regions and multiple contiguous genes, can be targeted. We have created single nucleotide changes in a YAC harboring the human beta-globin gene, a locus in which point mutations result in sickle cell disease and beta-thalassemias. We have also created SNPs in the human mdr1 (P-glycoprotein) gene in a yeast artificial chromosome. Our gene editing strategy can be used to create point mutations in human genes, including mutations that are rare in the population and difficult to obtain by other means. Panels of genes with custom SNPs/mutations have the added advantage of being isogenic, differing from each other only at the targeted site. These collections can be used for studies of gene function and can also serve as invaluable quality control materials for genetic diagnostic tests.