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， 使得能够进行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，事件直接影响下游生物医学研究。 然而，虽然双等位基因敲除的产生已经成为一个高效的过程，但我们的能力仍然是有限的。 有效地产生双等位基因敲入仍然受到严重限制。主要原因是，此外， 对于Cas介导的核酸内切裂解事件，需要克服以下障碍：首先， 同源重组需要在两个等位基因上发生。第二种是单一DNA修复模板 难以在单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 当使用两种DNA修复模板时，可以选择双等位基因编辑的细胞，但总体上， 效率进一步降低。 因此，迫切需要能够实现有效的双等位基因编辑的新技术， 强大的选择策略。我们假设，这一关键需求可以通过协同解决 使用Cas酶结合DNA重组和布尔逻辑与门。 基于这些想法，我们建议开发一个新的基因组编辑平台，重点关注两个特定的基因组编辑平台。 目的：（1）利用单一DNA修复模板进行双等位基因基因编辑和DNA重组 以及（2）开发构成布尔逻辑与门的分裂报告子系统， 可以高效地选择双等位基因编辑的克隆。 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测序的能力。 编辑和整合生物门到这个过程中，从而产生广泛和直接的影响， 在生物医学科学中。

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.