![]() Two factors are important for the function of sgRNA promoter activity and the specificity of the sgRNA. 2013), then directs the Cas nuclease to cleave the target sequence. SgRNA cassette: sgRNA is a programmable 20-nucleotide (nt) sequence that recognizes the target DNA sequence and an invariant scaffold sequence (Ran et al. The expression of Hpt is usually driven by a strong constitutive promoter, such as maize Ubiquitin 1 ( ZmUbi1), rice ACTIN 1, or Cauliflower mosaic virus ( CaMV) 35S, for the ubiquitous expression of the antibiotic-tolerance gene (Mikami et al. Plant phosphomannose isomerase can also be used as a selectable marker for rice transformation (Hu et al. Among these genes, Hpt is the most widely used selection marker which confers tolerance to the herbicide hygromycin because several crops have a natural tolerance to kanamycin. Selection cassette: In monocots, several genes have served as useful selection markers for the efficient selection of transgenic plants, such as neomycin- phosphotransferase ( NPTII), bar, mutated acetolactate synthase ( ALS), plant phosphomannose isomerase, and hygromycin phosphotransferase ( Hpt) (Miki and McHugh 2004 Hu et al. Plasmids used for the stable genome editing of plants require a selection cassette, known as a sgRNA cassette, and a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) cassette in the T-DNA region, while the selection cassette is not necessary for transient transformations (Fig. For transient transformations, particle bombardment and polyethylene glycol (PEG)-mediated methods are used to deliver plasmids or ribonucleoproteins (RNPs) into the plant cells (Woo et al. For stable transformations, the Agrobacterium-mediated transformation method is typically used to deliver transfer DNA (T-DNA) into the plant cell, where it is then inserted into the plant genome (Mikami et al. Genome-edited plants can be generated either by stable or transient transformation. To perform CRISPR/Cas9-mediated genome editing, the Cas9 endonuclease is guided by a single guide RNA (sgRNA) to recognize the complementary sequence and create double-strand breaks (DSBs), thereby generating a short deletion or insertion. The CRISPR/Cas9 system has been successfully used for genome editing in a variety of monocots, including rice ( Oryza sativa), wheat ( Triticum sp.), barley ( Hordeum vulgare), and maize ( Zea mays) (Feng et al. We have aimed to encompass a full spectrum of information for genome editing 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. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. 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. 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. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. 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. Currently, various vectors and online tools are available to aid sgRNA design. 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. 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. 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.
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