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

通过杂交两个不同的品系，每个品系都有自己独特的品质(和问题)，然后从它们的后代中获得结合了两个祖先的良好特征的植株，同时消除了一个或另一个亲本品系特有的任何遗传缺陷，从而开发出新的作物品种。编码这些有益或有害特征的基因实际上是在染色体上以线性序列携带的。当一个基因的有益版本与同一染色体上的有害特性非常接近时，好的和坏的特性几乎总是一起遗传的--减少了“好”特性的好处，因为有害的特性总是伴随着它一起遗传的。“CRISPR”技术允许遗传学家在几乎任何选定的位置切割染色体。该项目的目标是确定CRISPR是否可以用来分离紧密排列的有益和有害基因版本，有效地分离这两个特征，并允许它们单独遗传。CRISPR诱导的DNA双链断裂通过各种宿主编码的DNA修复途径进行修复，每条途径都产生不同类别的修复产物。直接端到端连接(NHEJ)被认为在很大程度上是无错误的，微同源介导的连接(MMEJ)会产生缺失和插入，而基于同源的重组修复(HR)，它使用一个未受损的模板(如同源染色体、姐妹染色单体或同源转基因)来取代邻近和包括断裂位置的核苷酸，可以导致靶向等位基因替换或染色体臂交换。本项目的目的是确定是否可以通过消除KU依赖的端到端连接途径和/或DNA聚合酶theta(TEB)介导的微同源介导的末端连接途径来提高拟南芥中CRISPR诱导的断裂基于同源修复的速率。第二个目标是确定基于CRISPR的拟南芥突变是否需要DNA聚合酶theta。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.