The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.

突破性的CRISPR（聚类的规则间隔的短回文重复序列）/ Cas9介导的基因组编辑技术在单子叶植物研究方面取得了巨大进展。但是，有效实施该技术需要考虑几个因素。为了生成基因组编辑的农作物，将单个向导（sg）RNA和Cas9 DNA传递到植物细胞中并进行表达，然后预测目标位置。对成功的靶向突变的分析表明，sgRNA和Cas9的表达水平，表达时机以及变体都需要精密调节。因此，这些基因的启动子和靶位点位置是基因组编辑效率的关键因素。当前，各种载体和在线工具可用于辅助sgRNA设计。此外，为减少原间隔子相邻基序（PAM）的序列限制，并出于其他目的，许多Cas蛋白变体和碱基编辑器可用于植物。因此，在植物稳定转化之前，对载体和靶位点的评估非常重要。此外，Cas9-sgRNA核糖核蛋白（RNPs）的交付是一种可用于防止sgRNA和Cas蛋白表达的转基因问题的策略。RNP可用于有效地产生无转基因的基因组编辑的作物，可减少与转基因生物的产生有关的转基因问题。在这篇综述中，我们介绍了用于单子叶植物中基因组编辑和鉴定无标记基因组编辑突变体的新技术。涵盖了四个主题：单子叶植物中用于基因组编辑的质粒的设计和构建；SpCas9的替代品；原生质体和CRISPR; 和筛选无标记的CRISPR / Cas9诱导的突变体。我们旨在涵盖用于单子叶植物基因组编辑的全部信息。