Abstract
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.
Highlights
The breakthrough clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome-editing technology has led to great progress in monocot research; several factors need to be considered for the efficient implementation of this technology
Multiplex genome editing can be achieved by the simultaneous delivery and expression of multiple single guide RNA (sgRNA); since most CRISPR/Cas9 components are transferred into plants via Agrobacterium-mediated transformations, an efficient plasmid construction strategy is required
Protoplasts are often used for plant science investigations (Marx 2016), and the convenience and speed of their transfection means they are an attractive model in which to assess the mutagenesis efficiency of a CRISPR/Cas system, including the validation of Cas protein codon optimizations or modifications, sgRNA target sites, the promoters used for sgRNA and Cas9 proteins, and different vector designs (Andersson et al 2017; Butt et al 2017; Cermak et al 2017; Endo et al 2019; Hsu et al 2019; Hsu et al in preparation; Li et al 2013; Li et al 2018; Liang et al 2014; Lowder et al 2015; Shan et al 2013; Sun et al 2015; Zong et al 2017, 2018)
Summary
The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; several factors need to be considered for the efficient implementation of this technology. The specificity of the sgRNA for its target DNA sequence is another factor that affects the efficiency of genome editing.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
