Targeted Genomic Integration by Engineered Transposon and CRISPR/Cas9 Components

通过工程转座子和 CRISPR/Cas9 组件进行靶向基因组整合

• 批准号: 504353267
• 负责人: Professor Dr. Zoltan Ivics
• 金额: --
• 依托单位: Institut für Transfusionsmedizin und Gentherapie (ITG)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/504353267?language=en
• 关键词: Targeted; Genomic; Integration; Engineered; Transposon

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.

理想的基因转移工具将能够以有效和位点特异性的方式进行治疗性核酸的染色体整合，而不会引入可能导致潜在严重副作用的靶基因组的不希望的改变。目前可用的技术都没有满足这一要求。包括CRISPR/Cas9系统在内的设计核酸酶在其作用中具有高度特异性，但依赖于将双链断裂（DSB）引入基因组中以促进转基因在所需靶标处的整合。这是基因组可能经历的最危险的DNA损伤形式之一。此外，DSB非预期脱靶引入基因组是一个重大的安全性问题。另一方面，整合基因递送载体，包括病毒和转座子，具有插入其遗传货物而不破坏靶基因组的优点。然而，整合以半随机方式发生，从而存在细胞基因的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，以实现高效和精确的整合治疗性转基因在一个DSB-免费的方式。我们将把CRISPR/Cas9的组成部分整合到非病毒的睡美人（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，其关于将SB转座事件靶向到满足由向导RNA（gRNA）指定的基因组安全港标准的多拷贝靶标和单拷贝染色体靶标中的效力。我们将探索一种新的SB转座酶突变体，该突变体在特异性方面表现出显着的增益，与gRNA介导的靶向预先选择的基因组位点相结合。最后，我们将设计SB转座酶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修复因子组成的融合蛋白，这些因子通过同源重组促进转基因整合，而Cas9衍生的切口酶仅切割靶DNA的一条链，因此将减轻对DSB的需求。最后，我们将评估最有前途的实验系统在治疗相关的人类T淋巴细胞的性能。将转基因有效定点整合到人类基因组安全区域的技术将显著有助于全面改善人类应用中基因转移的安全性。