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更接近修复人类患者缺陷基因的梦想。在过去的20年里，历代的可编程核酸酶技术都被应用到这个问题上。从锌指核酸酶，到TALEN，再到CRISPR-CAS-核酸酶，这类系统的灵活性有增加的趋势。其中几个系统已经被改造成使用嵌入在这些系统的第一个版本中的核酸酶的替代效应器部分，允许通过对染色体DNA的更好的耐受性和更可控的改变来修饰靶细胞。然而，缺乏一种灵活而有效的方法，在不产生双链(DS)断裂的情况下在定义的基因组位置引入新的遗传物质。如果不能迅速修复，这种DS断裂是致命的，它们也被认为是对基因组完整性的威胁，因此很容易在靶细胞中引发程序性细胞死亡。铅笔生物科学已经创建并正在开发一种全合成基因组调制系统(ApGet)，并寻求资金使该系统能够在关键靶点高效地敲入供体DNA序列。ApGet系统体积小(~0.7kb)，模块化，组成无CRISPR。它能够通过RNA引导将不同的效应蛋白招募到宿主基因组的特定位置。铅笔计划用其他成分取代ApGet的一些成分，允许通过一种根本不同的机制进行敲入，这种机制不会导致dsDNA断裂。其目的是招募捐赠者DNA到感兴趣的目标位置，以便提供安全、耐受性好和高效的敲入编辑，用于基因修复和产生工程细胞疗法。从长远来看，基因编辑可能对减轻遗传病的负担做出重大贡献，仅在英国就有300多万人受到影响。这些疾病中的许多最好的治疗方法是“一次即可完成”的治疗，其中包括以敲门为中介的等位基因缺陷部分的替换。

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.