Novel, synthetic gene editing technology for gene knock-ins without DNA cleavage

基因组编辑领域的最新进展使我们离修复人类患者缺陷基因的梦想更近了一步。在过去的20年里，连续的可编程核酸酶技术已经应用于这个问题。从锌指核酸酶，到TALENs，再到CRISPR- Cas核酸酶，这类系统的灵活性一直在增加。一些系统已经适应使用替代的效应片段来代替这些系统的第一个版本中嵌入的核酸酶，允许通过对染色体DNA的更好耐受和更可控的改变来修饰靶细胞。然而，目前缺少的是一种灵活而有效的方法，既能在基因组的特定位点引入新的遗传物质，又不会造成双链断裂。如果不能迅速修复，这种断裂是致命的，它们也被认为是对基因组完整性的威胁，因此很容易引发靶细胞的程序性细胞死亡。Pencil Biosciences已经创建并正在开发一种完全合成的基因组调节系统（ApGet），并正在寻求资金来改造该系统，以便在关键靶点上对供体DNA序列进行高效的敲入。ApGet系统体积小（约0.7 kb），模块化和非crispr组成。它能够通过rna引导将多种效应蛋白招募到宿主基因组的特定位点。Pencil计划将ApGet的部分组件替换为其他组件，允许通过一种根本不同的机制进行敲入，而不会导致dsDNA断裂。其目的是招募供体DNA到感兴趣的目标位点，以便为基因修复和产生工程细胞疗法提供安全、耐受性良好和高效的敲入编辑。从长远来看，基因编辑可能会对减轻遗传疾病的负担做出重大贡献，仅在英国就有300多万人受到遗传疾病的影响。许多这些疾病最好的治疗方法是“一了百了”的治疗，包括敲入介导的等位基因缺陷部分的替代。

Recent advances in the field of genome editing have moved us closer to the dream of repairing defective genes in human patients. Over the last 20 years, successive dynasties of programmable nuclease technologies have been applied to this problem. From zinc-finger nucleases, through TALENs to CRISPR--Cas- nucleases there has been a trend towards increasing flexibility of such systems. Several of the systems have been adapted to use alternative effector moieties to the nucleases embedded in the first versions of these systems, allowing target cell modification via better tolerated and more controllable changes to chromosomal DNA.What is missing however, is a flexible and effective method for introducing new genetic material at a defined locus of a genome without creating double-strand (ds) breaks. Such ds breaks are lethal if not rapidly repaired, and they are also recognized as a threat to genome integrity and hence can easily trigger programmed cell death in target cells.Pencil Biosciences has created, and is developing, a fully-synthetic genome modulation system (ApGet) and seeks funding to adapt the system to deliver highly-efficient knock-in of donor DNA sequences at key target sites. The ApGet system is small in size (~0.7 kb), modular and non-CRISPR in composition. It is capable of recruiting diverse effector proteins to specific sites in the host genome by means of RNA-guides.Pencil plans to replace some of ApGet's components with others allowing knock-ins via a fundamentally different mechanism that does not induce a dsDNA break. The aim is to recruit donor DNA to target sites of interest in order to deliver safe, well-tolerated and highly efficient knock-in edits for gene repair and the generation of engineered cell therapies.In the long term gene editing may make a significant contribution to reducing the burden of genetic diseases, which in the UK alone affect more than 3 million people. Many of these diseases would be best treated with a 'one and done' treatment involving a knock-in-mediated replacement of the defective section of an allele.