CRISPR/Cas9-mediated genome editing in Brassica

CRISPR/Cas9 is a valuable tool for both basic and applied research that has been widely applied to different plant species. In this chapter, we reviewed the application of CRISPR/Cas9 genome editing toolkit in Brassica crops. We also provided a case study in Brassica napus. Collectively, our results demonstrate that CRISPR/Cas9 is an efficient tool for creating targeted genome modifications at multiple loci in B. napus. These findings open many doors for biotechnological applications in oilseed crops.

Double strand break (DSB) repair pathways in plants and their application in genome engineering

In genome engineering, after targeted induction of double strand breaks (DSBs) researchers take advantage of the organisms’ own repair mechanisms to induce different kinds of sequence changes into the genome. Therefore, understanding of the underlying mechanisms is essential. This chapter will review in detail the two main pathways of DSB repair in plant cells, non-homologous end joining (NHEJ) and homologous recombination (HR) and sum up what we have learned over the last decades about them. We summarize the different models that have been proposed and set these into relation with the molecular outcomes of different classes of DSB repair. Moreover, we describe the factors that have been identified to be involved in these pathways. Applying this knowledge of DSB repair should help us to improve the efficiency of different types of genome engineering in plants.

Targeted modification of promoters

Although most genome editing efforts focus on modifications to gene coding regions, this chapter emphasizes genome editing of the upstream regulatory regions. Thoughtful editing of the promoter region will ultimately lead to improved plants, modified for more precise control of the intensity and specificity of native gene expression. In this chapter, we present an overview of the promoter or upstream regulatory region of a gene, and describe how this sequence is defined and studied. We then describe how the composition and arrangements of cis-regulatory elements within the promoter and the leading intron associated with the promoter region have been studied using classical transgenic approaches to reveal what regulatory components might be suitable for genome editing approaches. Finally, we offer some suggestions for pursuit of promoter editing and gene expression modulation, which will eventually lead to modified plants with an altered regulation of native gene expression.

The regulation of genome-edited crops

To explore the regulation of genome edited crops, this chapter describes the regulatory systems adopted by different countries as well as how the Cartagena Biosafety Protocol might apply. It finds there is a continuum of oversight, with some countries applying their GMO biosafety laws to all genome edited crops and other countries exempting certain genome edited techniques and/or products from any special oversight. For countries with what on the surface seem like similar regulatory policies, when their regulations are applied to identical products, they often produce different results, which will make international harmonization difficult. In addition, how much oversight to impose on different crops tends not to be based on scientific evidence. Finally, the regulatory treatment for multi-edited products is unclear. It will take time and additional the development of additional genome edited crops before there is sufficient clarity on how all types of genome edited crops will be regulated.

Genome editing of woody perennial trees

This chapter discusses the challenges facing woody perennial trees, namely the heterozygosity of outcrossing species and limited genomic resources. Gene knockouts (KO) represent the predominant applications of the CRISPR technology in woody perennial crops to date and will be the focus of this chapter. The issues considered for gene KO are generally applicable to other CRISPR applications targeting regulatory sequences or non-coding genes. Case studies from the Populus 4-coumarate:CoA ligase (4CL) family are presented to demonstrate the power of CRISPR in elucidating functional redundancy as well as specificity of duplicated genes in a perennial woody species.

Using TALENs for genome editing in plants

Transcription activator-like effector nucleases (TALENs) are powerful tools for precise and efficient locus-specific editing. Among the various genome editing tools that were developed prior to the CRISPR/Cas system, TALENs have been the most widely used, and they remain worthy of consideration due to their unique targeting properties and the current intellectual property landscape, which give them distinct advantages over CRISPR/Cas for some applications. This chapter briefly reviews and discusses use of TALENs in plants including their important distinguishing features, as well as design principles and tools, methods for construct assembly, and other available resources to assist researchers using this technology.

Advances in the generation of insertion-based genome edits in plants

Tremendous progress has been achieved in the field of gene editing in plants, such as with the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Because of the potential advantages associated with mutant creation and crop germplasm innovation, genome editing technology has been rapidly developed and widely used in crop improvement in recent years. In this review, we aim to document some of the important recent developments and applications of genome-editing tools, especially with respect to gene knock-ins. We introduce the mechanism underlying knock-ins and different outcomes of insertion. We also discuss genome editing tools and methods developed to improve insertion efficiencies. Additionally, we review the recent trends in genetic editing biotechnologies; several strategies are being developed to further improve the efficiency of plant gene knock-ins. Undoubtedly, CRISPR/Cas technology will boost the development of new plant breeding techniques tremendously.

Progress in precise and predictable genome editing in plants with base editing

Base editors are gene editing tools that allow targeted nucleic acid conversions, most commonly C>T and A>G, through pairing of deamination domains with impaired nucleases. Multiple deaminase domains and architectures have been demonstrated in planta across a wide array of species, with both cytosine and adenine base editing frequencies being observed at over 80%. The ability of base editors to introduce nucleic acid diversity while maintaining the same reading frame should make them powerful tools for plant genetic editing moving forward.

Advances in guide RNA design for editing plant genomes using CRISPR-Cas systems

This chapter discusses the general rules for selecting target sites for genome editing using the CRISPR-Cas technology and summarizes the bioinformatic tools that can be used to design sgRNA sequences.

Advances in assembling gRNA/Cas9 constructs in genome editing of plants

This chapter reviews the principles of CRISPR cloning in binary vectors and the different methods and elements employed, including the nucleases alternative to Cas9. It pays special attention to modular cloning strategies and multiplexing tools as well as the engineering of expanded Cas activities. Finally, the chapter includes a case study of the cloning of a nine gRNA multiplex construct and the analysis of its transformants in tobacco plants.