DNA Replicons for Plant Genome Engineering  

Abstract

Sequence-specific nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of plant genomes remains challenging due to ineffective methods for delivering reagents for genome engineering to plant cells. Here, we use geminivirus-based replicons for transient expression of sequence-specific nucleases (zinc-finger nucleases, transcription activatorlike effector nucleases, and the clustered, regularly interspaced, short palindromic repeat/Cas system) and delivery of DNA repair templates. In tobacco (Nicotiana tabacum), replicons based on the bean yellow dwarf virus enhanced gene targeting frequencies one to two orders of magnitude over conventional Agrobacterium tumefaciens T-DNA. In addition to the nuclease-mediated DNA doublestrand breaks, gene targeting was promoted by replication of the repair template and pleiotropic activity of the geminivirus replication initiator proteins. We demonstrate the feasibility of using geminivirus replicons to generate plants with a desired DNA sequence modification. By adopting a general plant transformation method, plantlets with a desired DNA change were regenerated in <6 weeks. These results, in addition to the large host range of geminiviruses, advocate the use of replicons for plant genome engineering.

Gene Targeting by Homology-Directed Repair in Rice Using a Geminivirus-Based CRISPR/Cas9 System

“…Upon infection, they produce numerous replicons through rolling-circle replication which may serve as repair templates during HDR (Baltes et al, 2014;Hanley-Bowdoin et al, 2013). The bean yellow dwarf virus (BeYDV) has been used in tobacco and tomato for this purpose, increasing KI efficiency 10 to 100-times compared to conventional T-DNA delivery (Baltes et al, 2014;Cermak et al, 2015).…”

“…The bean yellow dwarf virus (BeYDV) has been used in tobacco and tomato for this purpose, increasing KI efficiency 10 to 100-times compared to conventional T-DNA delivery (Baltes et al, 2014;Cermak et al, 2015). The wheat dwarf virus (WDV) infects a variety of grasses including most cereals and has been previously used to express foreign proteins in wheat and maize cells (Suarez-Lopez and Gutierrez, 1997).…”

“…To facilitate homologous recombination, the GFP-2A-NPTII cassette was flanked by two homology arms (~500 bp) for ACT1 or GST resulting in ~2.6-kb donor DNA fragments which were inserted into the WDV-GFP vector replacing the GFP expression cassette (designated as WDV2-ACT1 and WDV2-GST, respectively) ( Figure 1E). Previous studies have shown that CRISPR/Cas9-induced DSBs at the target site greatly increase the KI frequency (Baltes et al, 2014;Cermak et al, 2015).…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…The newly synthesized ssDNA can be converted into dsDNA and serve as template for the repair of the CRISPR-induced DBSs. Moreover, the viral Rep protein has a pleiotropic activity that enhances gene targeting potentially by interacting with host proteins affecting levels of HR [93]. The CRISPR elements and the donor cassette are usually integrated with the trans-acting viral polymerase Rep/RepA, the LIR and the short intergenic region (SIR) into a single plasmid to coordinate the expression of the CRISPR-Cas system and the production of a high copy number of the repair template.…”

“…In theory, the BeYDV replicon could be used to mediated gene knock-in in other dicotyledonous species provided that they belong to its host range, like Arabidopsis thaliana, tobacco, Datura stramonium or French bean (Phaseolus vulgaris) (Liu et al, 1997). For monocots, other viruses from the same mastrevirus genius could be used, such as the wheat dwarf virus (WDV) or maize streak virus (MSV) [93]. Recently, a modified replicon from WDV carrying the CRISPR-Cas9 system and a repair template allowed to increase by 12-fold the efficiency of CRISPRinduced gene knock-in (5.85%) at an endogenous ubiquitin locus in wheat cells [94] (Table 1).…”