Plant genome editing with TALEN and CRISPR

Abstract

Genome editing promises giant leaps forward in advancing biotechnology, agriculture, and basic research. The process relies on the use of sequence specific nucleases (SSNs) to make DNA double stranded breaks at user defined genomic loci, which are subsequently repaired by two main DNA repair pathways: non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ can result in frameshift mutations that often create genetic knockouts. These knockout lines are useful for functional and reverse genetic studies but also have applications in agriculture. HDR has a variety of applications as it can be used for gene replacement, gene stacking, and for creating various fusion proteins. In recent years, transcription activator-like effector nucleases and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated protein 9 or CRISPR from Prevotella and Francisella 1 have emerged as the preferred SSNs for research purposes. Here, we review their applications in plant research, discuss current limitations, and predict future research directions in plant genome editing.

Risk associated with off-target plant genome editing and methods for its limitation

“…Their source may be native to the organism or to a near relative, or they may represent foreign DNA which is synthesized or transgenic. The nature of these various genome-editing outcomes in crops has been the subject of extensive reviews [11][12][13].…”

“…The choice of system relates to ease and breadth of use and the efficiency and specificity of editing. Both ZFNs and TALENs function as dimers with each monomer comprising a fusion protein-binding domain which together determine sequence specificity; cleavage is carried out by the attached FokI nuclease catalytic domain [13,21,22]. Successful applications of ZFNs and TALENs have been shown in mammalian cells, plant cells, and other organisms [17,[23][24][25][26][27].…”

“…Successful applications of ZFNs and TALENs have been shown in mammalian cells, plant cells, and other organisms [17,[23][24][25][26][27]. Unfortunately, because of low affinity and low specificity, gene editing with ZFNs has displayed high frequencies of off-target edits and high toxicity [13]. The design of TALENs involves one-to-one recognition rules between protein repeats and nucleotide sequences, which results in improved editing efficiency and reduced off-target effects [21,28].…”

“…It is difficult, however, to construct the nuclease protein and a new TALEN protein must be generated for each DNA target site, which increases time and costs for development [29]. Additionally, the cut efficiency for TALENs varies depending on target sequence and TALENs cannot target methylated DNA [13]. As we know, DNA methylation of plant genome controls plant growth, development, transcription, DNA repair, gene expression, and cell differentiation, and is particularly involved in specific gene silencing.…”

“…Both DNA methylation and hypermethylation contribute to the adaptation of plants to stress and stable inheritance [30][31][32], and thus, TALENs have limited application for these types of breeding goals. Compared with ZFNs and TALENs, the CRISPR/Cas9 system exhibits greater simplicity, efficiency, and versatility, and it also can target methylated DNA [13,33]. CRISPR/Cas9 has been widely adopted for genome editing in plants.…”