How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis

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.

Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation

“…Heterologous protein expression in plants is achieved by stable or transient transformation by delivery of genes of interest harbored in Agrobacterium T-DNA 74 . AMT has emerged as a vehicle for the application of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing of plants 75 . Genome-editing reagents are delivered to plants mostly through a stable transformation that requires segregation of Cas9 by Mendelian segregation to achieve transgene-free genome-edited plants (null segregants) 43 .…”

Genome Editing and Protoplast Regeneration to Study Plant–Pathogen Interactions in the Model Plant Nicotiana benthamiana

“…In fact, unexpected mutations can occur in any tissue culture process and even under natural conditions (Lin and Chang, 1998 ; Yue et al, 2020 ). In crop breeding, even if mutations occur, desired offspring can be identified through selection from a wide range of gene-edited regenerated plants, without the ethical problems associated with the human application of genome editing (Tang et al, 2019 ).…”

“…We chose to address this problem by using the powerful genome-editing tool CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPRassociated protein). CRISPR-Cas core technology involves programmable DNA cleavage by the Cas protein at DNA sites specified by the targeting sequence in a guide RNA (gRNA; review by Yue et al, 2020). The use of CRISPR-Cas has greatly accelerated plant research and crop breeding in recent years (Li et al, 2013;Nekrasov et al, 2013;Shan et al, 2013;Li and Xia, 2020;Yue et al, 2020).…”

“…We chose to address this problem by using the powerful genome-editing tool CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein). CRISPR-Cas core technology involves programmable DNA cleavage by the Cas protein at DNA sites specified by the targeting sequence in a guide RNA (gRNA; review by Yue et al, 2020 ). The use of CRISPR-Cas has greatly accelerated plant research and crop breeding in recent years (Li et al, 2013 ; Nekrasov et al, 2013 ; Shan et al, 2013 ; Li and Xia, 2020 ; Yue et al, 2020 ).…”

Understanding Wheat Starch Metabolism in Properties, Environmental Stress Condition, and Molecular Approaches for Value-Added Utilization

“…In the case of plants, since ethical issues are somewhat insignificant, flexible regulation should be carried out. Moreover, transgene-free genome-edited plants can be easily generated by ribonucleoproteins (RNP) or Mendelian segregation [196,197]. Therefore, if policy and governance issues are addressed at national and international levels, plant genome editing can play a key role in developing useful crops, along with rapid scientific progress.…”