Gene editing of a human gene in yeast artificial chromosomes using modified single-stranded DNA and dual targeting.
A single-nucleotide polymorphism (SNP) in a human gene can alter the behavior of the corresponding protein, and thereby affect an individual's response to drug therapy. Here, we describe a novel dual-targeting approach for introducing an SNP of choice into virtually any gene, through the use of modified single-stranded oligonucleotides (MSSOs). We use this strategy to create SNPs in a human gene contained in a yeast artificial chromosome (YAC). In the dual-targeting protocol, two different MSSOs are designed to edit two different bases in the same cell. A change in one of these genes is selective while the other is non-selective. We show that the population identified by selective pressure is enriched for cells that bear an edited base at the nonselective site. YACs with human genomic inserts containing particular SNPs or haplotypes can be used for pharmacogenomic applications, in cell lines and in transgenic animals.
NaPro BioTherapeutics, Genomics Division, Newark, DE, USA. ekmiec@udel.edu
A J
AJ
van Brabant
J K
JK
Williams
H
H
Parekh-Olmedo
E B
EB
Kmiec
D001483:Base Sequence; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017393:RNA Editing
D004277:DNA, Single-Stranded