Abstract

The conversion of bacterial CRISPR/Cas defense system into a simple and efficient tool for genome manipulations brought experimental biology into new dimensions. Suddenly, genome editing reached many groups most of which were interested in it but not able to employ the available time- and labor-consuming approaches of the pre-CRISPR era. In plant biology and biotechnology, CRISPR/Cas gene editing became the second most important technology after plant transformation. Actually, it relies on the available array of methods of gene delivery. While sufficient for most purposes, the classic gene transfer methods might become a problem for some experimental settings. The main obstacle is that they include DNA delivery and, frequently, its subsequent integration into cellular genome. For this reason novel methods to achieve gene editing without the need of stable transformation and even without DNA delivery were developed. These new approaches include in vitro ribonucleoprotein complexes formulations (delivered by microinjection, particle bombardment, electroporation, liposomes etc.), use of virus-like particles and employment of bacterial secretory systems for Cas/gRNA delivery. The first attempts to achieve DNA-free editing were made less than ten years ago. Later, different types of animal and plant cells were addressed. In this mini review we try to summarize the current developments and emerging trends in the field of DNA-free editing in plants.

摘要

将细菌CRISPR / Cas防御系统转换为用于基因组操作的简单有效的工具，将实验生物学带入了新的领域。突然，基因组编辑进入了许多小组，其中大多数对此感兴趣，但无法采用CRISPR之前时代可用的耗时且费力的方法。在植物生物学和生物技术中，CRISPR / Cas基因编辑成为继植物转化后第二重要的技术。实际上，它依赖于可用的一系列基因传递方法。尽管对于大多数目的已经足够了，但是经典的基因转移方法可能会在某些实验环境中成为问题。主要障碍是它们包括DNA传递，通常包括随后的整合到细胞基因组中。因此，开发了不需要稳定转化甚至不需要DNA递送即可实现基因编辑的新方法。这些新方法包括体外核糖核蛋白复合物制剂（通过显微注射，粒子轰击，电穿孔，脂质体等进行递送），使用病毒样颗粒并利用细菌分泌系统进行Cas / gRNA递送。不到十年前就进行了无DNA编辑的首次尝试。后来，研究了不同类型的动植物细胞。在本微型综述中，我们尝试总结植物中无DNA编辑领域的当前发展和新兴趋势。