A tool for precise, target-specific, efficient and affordable genome editing, it is a dream for many researchers, from those who do basic research to those who use it for applied research. Since 2012, we have the tool that almost fulfils such requirements; it is based on CRISPR/Cas systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight the key factors affecting its mutagenesis outcomes. Firstly, factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state or Cas9 variant, and how long it remains in place after cleavage. Secondly, factors affecting the follow-up DNA repair mechanisms include mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.

中文翻译：

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如何在植物中使用 CRISPR/Cas9 - 从靶位点选择到 DNA 修复

它是一种精确、针对特定目标、高效且经济实惠的基因组编辑工具，是许多研究人员的梦想，从基础研究到应用研究。从2012年开始，我们就有了几乎满足这样要求的工具；它基于 CRISPR/Cas 系统。然而，即使 CRISPR/Cas 也存在一些可能令用户感到惊讶的局限性和障碍。在这篇综述中，我们重点关注最常用的变体，即来自化脓性链球菌的 CRISPR/Cas9，并强调影响其诱变结果的关键因素。首先，影响 CRISPR/Cas9 活性的因素，例如靶序列、染色质状态或 Cas9 变体的影响，以及切割后保持原位的时间。其次，影响后续DNA修复机制的因素主要包括细胞类型和细胞周期阶段，但也包括例如Cas9切割产生的DNA末端的类型（平端/交错）。此外，我们注意到在植物、酵母和动物中使用 CRISPR/Cas9 之间存在一些差异，因为来自各个王国的知识不能完全转移。对这些因素的认识可以增加实现植物基因组编辑预期结果的可能性，为此我们提供了详细的指南。