Abstract
DNA-dependent protein kinase (DNA-PKcs) is essential for DNA repair pathways. The activity of DNA-PKcs is regulated by different phosphorylation sites in response to DNA double strand breaks (DSBs). It was reported that three alanine substitutions at the Thr2605 phosphorylation cluster, including Thr2605、Thr2634 and Thr2643, on exon 58 (DNA-PKcs3A/3A) would cause early bone marrow failure in homozygous mice. This study is aimed to generate DNA-PKcs3A/3A mutant mice in the BALB/c background and to evaluate its potential for humanization. A previous attempt by direct injection of Cas9 plasmid, guide RNA, and donor DNA into BLAB/c fertilised eggs failed to generate the desired mutant. Thus an alternative approach by combining CRISPR/Cas9 and neo-based screening on BALB/c embryonic stem cells is taken. Specifically, we use Cas9 and exon 58-targeting guide RNAs to induce DNA breaks in BALB/c embryonic stem cells. At the same time we supply a mutant template of DNA-PKcs3A within a cassette that has the PGK:Neo selectable marker for positive selection and two flanking arms for homologous recombination. In the future, the selected DNA-PKcs3A mutant knock-in ES cells will be injected into blastocysts to produce chimeric mice by foster mothers. Hopefully these mutant mice will have the ability of germline transmission to generate a line of homozygous DNA-PKcs3A/3A offsprings after further breeding. These mice will be evaluated whether they can serve as a better platform for humanization by human CD34+ cells. Human CD34+ cells are hematopoietic stem cells that have been known to be able to establish the human immune system in immune compromised mice. With expected early bone marrow failure and immune deficiency in homozygous DNA-PKcs3A/3A mice, we hope these properties could augment the performance of CD34+ cell-based humanization and serve the need of an excellent platform for establishing the human immune system. Once succeeded, my work will have great contributions to drug testing and disease research that require faithfulness to bona fide human immunity.