PDF(Pubmed)
Abstract：
Non-viral DNA donor template has been widely used for targeted genomic integration by homologous recombination (HR). This process has become more efficient with RNA guided endonuclease editor system such as CRISPR/Cas9. Circular single stranded DNA (cssDNA) has been harnessed previously as a genome engineering catalyst (GATALYST) for efficient and safe targeted gene knock-in. Here we developed enGager, a system with enhanced GATALYST associated genome editor, comprising a set of novel genome editors in which the integration efficiency of a circular single-stranded (css) donor DNA is elevated by directly tethering of the cssDNA to a nuclear-localized Cas9 fused with ssDNA binding peptides. Improvements in site-directed genomic integration and expression of a knocked-in DNA encoding GFP were observed at multiple genomic loci in multiple cell lines. The enhancement of integration efficiency, compared to unfused Cas9 editors, ranges from 1.5- to more than 6-fold, with the enhancement most pronounced for transgenes of > 4Kb in length in primary cells. enGager-enhanced genome integration prefers ssDNA donors which, unlike traditional dsDNA donors, are not concatemerized or rearranged prior to and during integration Using an enGager fused to an optimized cssDNA binding peptide, exceptionally efficient, targeted integration of the chimeric antigen receptor (CAR) transgene was achieved in 33% of primary human T cells. Enhanced anti-tumor function of these CAR-T primary cells demonstrated the functional competence of the transgenes. The \'tripartite editors with ssDNA optimized genome engineering\' (TESOGENASE™) systems help address the efficacy needs for therapeutic gene modification while avoiding the safety and payload size limitations of viral vectors currently used for CAR-T engineering.
摘要：
非病毒DNA供体模板已广泛用于通过同源重组(HR)的靶向基因组整合。使用RNA引导的核酸内切酶编辑器系统(如CRISPR/Cas9),该过程变得更有效。环状单链DNA（cssDNA）先前已被用作基因组工程催化剂（GATALYST），用于有效且安全的靶向基因敲入。在这里我们开发了enGager，具有增强的GATALYST相关基因组编辑器的系统，包括一组新的基因组编辑器，其中通过将cssDNA直接连接到与ssDNA结合肽融合的核定位的Cas9来提高环状单链(css)供体DNA的整合效率。在多个细胞系中的多个基因组基因座处观察到编码GFP的敲入DNA的定点基因组整合和表达的改善。集成效率的提高，与未融合的Cas9编辑相比，范围从1.5倍到6倍以上，对于原代细胞中长度>4Kb的转基因,增强最为明显。enGager增强的基因组整合更喜欢ssDNA供体，与传统的dsDNA供体不同，使用与优化的cssDNA结合肽融合的enGager，异常高效，在33%的原代人T细胞中实现了嵌合抗原受体(CAR)转基因的靶向整合。这些CAR-T原代细胞的增强的抗肿瘤功能证明了转基因的功能能力。“具有ssDNA优化基因组工程的三方编辑器”（TESOGENASETM）系统有助于解决治疗性基因修饰的功效需求，同时避免了目前用于CAR-T工程的病毒载体的安全性和有效载荷大小限制。
