Targeted Genomic Integration by Engineered Transposon and CRISPR/Cas9 Components

理想的基因转移工具将能够以有效和特定部位的方式实现治疗性核酸的染色体整合，而不会导致目标基因组的不必要改变，这可能会导致潜在的严重副作用。目前可用的技术都不能满足这一要求。设计核酸酶，包括CRISPR/Cas9系统，在其作用上具有高度的特异性，但依赖于在基因组中引入双链断裂(DSB)来促进所需靶点的转基因整合。这是基因组可能经历的最危险的DNA损伤形式之一。此外，将DSB意外地非靶标导入基因组是一个重大的安全问题。另一方面，整合基因传递载体，包括病毒和转座子，具有插入其基因货物而不破坏目标基因组的优势。然而，整合以半随机的方式发生，因此存在细胞基因插入激活或失活的风险。在这个项目中，我们的目标是增强基因组工程工具，以无DSB的方式实现治疗性转基因的高效和精确整合。我们将把CRISPR/Cas9的组件整合到非病毒、睡美人(SB)转座子介导的基因整合框架中，以增加其特异性。我们将评估由催化失活的Cas9和Sb转座酶组成的融合蛋白的性能，看看它们将Sb转座事件靶向多拷贝靶和单拷贝染色体靶的有效性，这些靶满足GUIDE RNAs(GRNAs)指定的基因组安全港标准。我们将探索一种新的SB转座酶突变体，它结合gRNA介导的预先选择的基因组位置的靶向，在特异性方面显示出显著的提高。最后，我们将对SB转座酶进行改造，以降低其内在整合活性，从而在CRISPR/Cas9组件存在的情况下将平衡转向靶向插入。我们将设计由DNA修复因子和Cas9衍生的镍酶组成的融合蛋白，这些DNA修复因子通过同源重组促进转基因整合，而Cas9衍生的镍酶只切割目标DNA的一条链，因此将减少对DSB的需求。最后，我们将评估最有希望的实验系统在治疗相关的人类T淋巴细胞上的性能。将有效的、定点定向的转基因整合到人类基因组中的安全区域的技术将大大有助于全面改善人类应用中基因转移的安全状况。

An ideal gene transfer tool would enable chromosomal integration of therapeutic nucleic acids in an efficient and site-specific manner without introducing unwanted alterations of the target genome, which might lead to potentially severe side effects. None of the currently available technologies fulfill this requirement. Designer nucleases, including the CRISPR/Cas9 system, are highly specific in their action, but rely on introducing a double-strand break (DSB) into the genome to promote transgene integration at the desired target. This is one of the most dangerous forms of DNA damage the genome can experience. In addition, unintended off-target introduction of DSBs into the genome represents a significant safety concern. On the other hand, integrating gene delivery vectors, including viruses and transposons, have the advantage of inserting their genetic cargo without breakage of the target genome. However, integration occurs in a semi-random fashion, thereby presenting a risk of insertional activation or inactivation of cellular genes. In this project we aim at enhancing genome engineering tools to achieve highly efficient and precise integration of therapeutic transgenes in a DSB-free manner. We will incorporate components of CRISPR/Cas9 into the framework of non-viral, Sleeping Beauty (SB) transposon-mediated gene integration to increase its specificity. We will evaluate the performance of fusion proteins consisting of catalytically inactive Cas9 and the SB transposase with respect to their potency of targeting SB transposition events into multi-copy targets and single-copy chromosomal targets that satisfy the criteria of genomic safe harbors, specified by guide RNAs (gRNAs). We will explore a novel SB transposase mutant that displays a significant gain in specificity, in conjunction with gRNA-mediated targeting to pre-selected genomic sites. Finally, we will engineer the SB transposase to lower its intrinsic integration activity, thereby shifting the balance towards targeted insertions in the presence of CRISPR/Cas9 components. We will engineer fusion proteins composed of DNA repair factors that promote transgene integration through homologous recombination and Cas9-derived nickases that cut only one strand of the target DNA and therefore would alleviate the need for a DSB. Finally, we will evaluate the performance of the most promising experimental system in therapeutically relevant human T lymphocytes. Technologies for efficient, site-directed transgene integration into safe regions in the human genome would significantly contribute to an overall improvement of the safety profile of gene transfer in human applications.