EAGER: DNA polymerase theta and the processing of double strand breaks

University Of California-Davis, Davis CA

Investigators

Abstract

New varieties of crops are developed by crossing two different lines, each with their own, unique qualities (and problems) and then deriving plants from among their descendants that combine the good traits of both ancestors while at the same time eliminating any genetic deficiencies unique to one or the other parent line. The genes encoding these beneficial or harmful traits are physically carried in a linear series on chromosomes. When a beneficial version of a gene is located very close to a detrimental trait on the same chromosome, the good and bad traits will virtually always be inherited together- reducing the benefit of the “good” trait, as the harmful trait is always inherited along with it. “CRISPR” technology allows geneticists to cut a chromosome at virtually any chosen location. The goal of this project is to determine whether CRISPR can be used to separate closely-spaced beneficial and harmful versions of genes, effectively unlinking the two traits and allowing them to be inherited separately. CRISPR-induced DNA double strand breaks are repaired via a variety of host-encoded DNA repair pathways, each producing a different class of repair product. Direct end to end ligation (Nonhomologous end-joining, NHEJ) is thought to be largely error-free, microhomology-mediated joining (MMEJ) creates deletions and insertions, and homology-based recombinational repair (HR), which employs an undamaged template (such as a homologous chromosome, a sister chromatid, or a homologous transgene) to replace nucleotides adjacent to and including the site of the break, can result in targeted allelic substitution or chromosome arm exchange. The goal of this project is to determine whether the rate of homology-based repair of CRISPR induced breaks can be enhanced in Arabidopsis through the elimination of the KU-dependent end to end rejoining pathway and/or the DNA polymerase theta (TEB)-mediated microhomology-mediated end joining pathway. A second goal is to determine whether DNA polymerase theta is required for CRISPR-based mutagenesis in Arabidopsis. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.