Advanced Editing of the Nuclear and Plastid Genomes in Plants T ⁎ Agnieszka A
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Plant Science 273 (2018) 42–49 Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci ☆ Advanced editing of the nuclear and plastid genomes in plants T ⁎ Agnieszka A. Piateka, Scott C. Lenaghanb,c, C. Neal Stewart Jr.a, a Department of Plant Sciences, University of Tennessee, Knoxville, TN, 37996, USA b Department of Food Science, University of Tennessee, Knoxville, TN, 37996, USA c Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville, TN, 37996, USA ARTICLE INFO ABSTRACT Keywords: Genome editing is a powerful suite of technologies utilized in basic and applied plant research. Both nuclear and Synthetic biology plastid genomes have been genetically engineered to alter traits in plants. While the most frequent molecular CRISPR outcome of gene editing has been knockouts resulting in a simple deletion of an endogenous protein of interest Synplastome from the host’s proteome, new genes have been added to plant genomes and, in several instances, the sequence Metabolic engineering of endogenous genes have been targeted for a few coding changes. Targeted plant characteristics for genome Genome editing editing range from single gene targets for agronomic input traits to metabolic pathways to endow novel plant function. In this paper, we review the fundamental approaches to editing nuclear and plastid genomes in plants with an emphasis on those utilizing synthetic biology. The differences between the eukaryotic-type nuclear genome and the prokaryotic-type plastid genome (plastome) in plants has profound consequences in the ap- proaches employed to transform, edit, select transformants, and indeed, nearly all aspects of genetic engineering procedures. Thus, we will discuss the two genomes targeted for editing in plants, the toolbox used to make edits, along with strategies for future editing approaches to transform crop production and sustainability. While CRISPR/Cas9 is the current method of choice in editing nuclear genomes, the plastome is typically edited using homologous recombination approaches. A particularly promising synthetic biology approach is to replace the endogenous plastome with a ‘synplastome’ that is computationally designed, and synthesized and assembled in the lab, then installed into chloroplasts. The editing strategies, transformation methods, characteristics of the novel plant also affect how the genetically engineered plant may be governed and regulated. Each of these components and final products of gene editing affect the future of biotechnology and farming. 1. Introduction contrast, the plastid genome (plastome) is of prokaryotic nature. Its genes can be expressed from operons with untranslated regions playing Editing the genome housed in the plant nucleus, as well as the much an important role in gene expression. Foreign DNA is typically bom- smaller genome housed in the plastid have tremendous potential to barded into chloroplasts using the gene gun and integrated into a target accelerate crop improvement to meet global environmental and agri- locus in the plastome by homologous recombination (HR). The choice cultural challenges. Modern genetic engineering and sequencing tech- of genome for editing depends on the research or product. For instance, nologies have provided key insight on how DNA modification can im- single-gene encoded phenotypic traits (as resistance to herbicides), pact cellular functions and the subsequent phenotypes. Such achieved through knockout or knock-in are better suited for nuclear information is critical to understanding how we can improve crop engineering whereas plastomes are better targets for manipulating quality and yield to meet the growing global food production needs. metabolic pathways or high-level production of recombinant proteins. There are vast differences in the characteristics of nuclear and A transformational breakthrough in genome editing occurred with plastid genomes, which lead to different engineering and editing ap- the discovery of programmable nucleases (also called site-specific nu- proaches (Table 1). The nuclear genome of eukaryotes, including an- cleases, SSNs, or site-directed nucleases-SDNs) [1,2]. Meganucleases, giosperms, has eukaryotic gene architecture, regulation and processing. zinc finger nucleases (ZFNs), transcription activator-like effector nu- Most often, ‘foreign’ DNA is randomly inserted into the plant nuclear cleases (TALENs), and more recently, the clustered regularly inter- genome using Agrobacterium-mediated transformation. Monocistronic spaced short palindromic repeats (CRISPR)/CRISPR associated protein gene structure is the rule, i.e., each gene has its own promoter. In 9 (Cas9) have been used in crop molecular breeding. SSNs, in general, ☆ This article is part of a special issue entitled “Synthetic biology meets plant metabolism”, published in the journal Plant Science 273, 2018. ⁎ Corresponding author. E-mail address: [email protected] (C. Neal Stewart). https://doi.org/10.1016/j.plantsci.2018.02.025 Received 14 December 2017; Received in revised form 24 February 2018; Accepted 26 February 2018 Available online 03 March 2018 0168-9452/ © 2018 Elsevier B.V. All rights reserved. A.A. Piatek et al. Plant Science 273 (2018) 42–49 Table 1 A comparison of the characteristics between plant genomes housed in the nucleus and those housed in plastids from an genetic engineering perspective. The features of interest range from the differences in molecular genetics inherent in each genome to the particular constraints in available biotechnological and synthetic biology tools. Nuclear genome features Plastid genome features Targeted mutations introduced predominantly through NHEJ, or HR when an external DNA template Mutations and transgenes introduced at defined loci through the prevalent is provided HR mechanism Genes are structured as monocistrons, i.e., each gene has its own promoter and terminator A single promoter can drive the expression of one-to-several transgenes as an operon Low relative gene expression High transgene copy number leads to silencing and increase likelihood Very high expression is possible High transgene copy is certain with no of deleterious position effects Proteins can be targeted to a number of organelles—alternative silencing or position effects. Proteins accumulate only in plastids targeting is feasible Relatively quick estimates can be made for gene expression and protein targeting Currently there are no rapid transgene expression assays available Transformation performed predominantly via Agrobacterium, also PEG and biolistic mediated vector Transformation performed typically via biolistic vector delivery delivery Possible transgene escape via pollen Maternal inheritance in most species, i.e., no transgene flow via pollen have tremendous utility to introduce mutations in genomes at various the mutation is in a regulatory region (promoter, 5′ untranslated region efficiencies. Certainly CRISPR/Cas9 has emerged as a game changer in (UTR), and 3′UTR). crop molecular breeding as evidenced by step-changes of peer-reviewed papers in recent years using this technique [3]. 2.1. Genome editing or genome vandalism? By far, most efforts in plant genetic engineering, have focused on the nuclear genome; however, the mitochondrial and plastid genomes Typically, the use of SSNs is referred to as genome editing. But, are essential to plants, contain non-redundant genes and metabolic George Church refers to simple protein knockouts as “genome vand- pathways, and are much smaller. Arguably the most important of all alism” (Stat News: https://www.statnews.com/2016/04/20/clever- plastome genes codes for the Rubisco large subunit (rbcL). It is neces- crispr-advance-unveiled/), akin to ripping pages out of a book. sary for carbon fixation and is the most abundant protein on Earth. CRISPR-induced indel formation within the coding region typically Utilizing genome editing to increase carboxylation of Rubisco, while leaves a gene non-functional, thus, knock-out experiments are the most decreasing photooxidation would improve the carbon fixation effi- frequent use of genome “editing”. ciency and photosynthetic capacity, which, in turn, would drastically – increase crop yield [4 6]. 2.2. A brief history of SSN development and utilization in plants While SSNs have been used to genome edit in vitro human cell mitochondria [7], there are no reports on the use of this technology to Chronologically, meganucleases were the first platform used for target the plastid genome. One challenge in SSN-mediated genome site-specific genome editing. Meganucleases are natural DNA nucleases editing plastomes is reaching homoplasmy without transplastomic su- [12], and have been engineered to recognize user-defined DNA se- fl fi per uous DNA (e.g., selection markers or target sites for site-speci c quences and introduce mutations within the genome [13,14]. Specifi- recombinases) [8]. Despite the dearth of SSN research for plastid cally for plants, new meganuclease delivery systems have been estab- genome engineering, the possibility for large-scale production of lished via biolistics for nanoparticle delivery [15] and through RNA pharmaceuticals, industrial enzymes, biomaterials, and biofuels within viruses [16]. In contrast to meganucleases, ZFNs are chimeric nucleases plastids necessitates further research into this area. Thus, in this review that bind DNA through multiple zinc finger domains [17,18] and cut we explore recent advances in nuclear and plastid genome engineering, using