Enhancing homozygous genome editing through DNA recombination and biologic gates

项目摘要 随着CRISPR/CAS系统的发现，人类基因组工程发生了革命性的变化， 实现了大量的基因组编辑操作。我们控制人类基因组编辑的能力 因此，这些事件直接影响下游的生物医学研究。 然而，尽管双等位基因敲除的产生已经成为一个高效的过程，但我们的能力 要有效地产生双等位基因敲入仍然受到严重的限制。造成这一现象的主要原因是 对于CaS介导的内切核裂解事件，需要克服以下障碍：第一， 需要在两个等位基因上进行同源重组。第二个使用单个DNA修复模板 很难在单个细胞水平上可靠地区分单等位基因编辑和双等位基因编辑。第三，在案件中 在使用两个DNA修复模板的情况下，可以选择双平行编辑的细胞，但总体 效率进一步降低。 因此，迫切需要新技术来实现高效的双等位编辑以及 强大的选择策略。我们假设，这一关键需求可以通过协同作用来解决 利用CaS酶结合DNA重组和布尔逻辑与门。 基于这些思想，我们提出了开发一个新的基因组编辑平台，重点放在两个具体的 目的：(1)利用单个DNA修复模板和DNA重组来促进双等位基因的基因组编辑 以及(2)开发构成布尔逻辑与门的分离报告器系统，使得 可以高效率地选择双平行编辑的克隆。 这些新方法将显著提高我们进行高效双等位基因人类基因组分析的能力 编辑并将生物门整合到这一过程中，从而产生广泛和直接的影响 横跨生物医学科学。

Project Summary With the discovery of the CRISPR/Cas system, human genome engineering has been revolutionized, enabling a large number of genome editing operations. Our ability to control human genome editing events therefore directly affects downstream biomedical research. However, while the generation of biallelic knockouts has become a highly efficient process, our ability to efficiently generate biallelic knock-ins is still severely limited. Key reasons for this are that in addition to a Cas-mediated endonucleolytic cleavage event the following hurdles need to be overcome: First, homologous recombination needs to occur on both alleles. Second with a single DNA repair template it is difficult to reliably distinguish monoallelic from biallelic editing at a single cell level. Third, in cases where two DNA repair templates are used, biallelically edited cells can be selected but the overall efficiency is further reduced. There is hence an urgent need for new technologies that enable efficient biallelic editing together with powerful selection strategies. We hypothesize that this critical need can be addressed by synergistically employing a Cas enzyme in conjunction with DNA recombination and BOOLEAN logic AND gates. Based on these ideas we propose to develop a new genome editing platform, focusing on two specific aims: (1) To facilitate biallelic genome editing with a single DNA repair template and DNA recombination and (2) To develop a split reporter system that constitutes a BOOLEAN logic AND gate so that biallelically edited clones can be selected with high efficiency. These new approaches will significantly increase our ability to perform efficient biallelic human genome editing and integrate biologic gates into this process, thus having widespread and immediate impact across biomedical sciences.