Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants

Abstract

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines transcription activator-like effector nuclease (TALEN)-mediated induction of double strand breaks (DSBs) and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described.

The Impact of Plant-Parasitic Nematodes on Agriculture and Methods of Control

“…The use of site-speciic DNA endonucleases including Zinc inger nucleases (ZFNs), [161] transcription activator-like efector nucleases (TALENs) [162] and now clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 [163] have equipped researchers with the ability to speciically inactivate genes and target genetic regions for homologous recombination of input DNA. In general, double-stranded breaks introduced by nucleases activates DNA repair mechanisms which generate mutations in the target sequence conferring a loss of expression i.e., gene editing.…”

Applications of New Breeding Technologies for Potato Improvement

“…In another study, Butler et al ( 2016 ) utilized a geminivirus replicon (GVR) vector for delivering sequence-specific nucleases (SSNs) to target the potato herbicide tolerance gene ALS1 and regenerated transformants carrying a point mutation in ALS1 gene were confirmed for herbicide susceptibility. Forsyth et al ( 2016 ) reported the targeted integration of transgene into a pre-selective, transcriptionally active site of potato genome using TALEN system coupled with a molecular marker, i.e., the mutated Acetolactate synthase ( ALS ) gene. Potato Ubi7 (constitutively expressing gene) was selected as a target for TALEN and after its functional confirmation in a transient system ( N. benthamiana ), Agrobacterium -mediated transformation was used to develop transgenic potatoes ( S. tuberosum cv.…”

“…Potato Ubi7 (constitutively expressing gene) was selected as a target for TALEN and after its functional confirmation in a transient system ( N. benthamiana ), Agrobacterium -mediated transformation was used to develop transgenic potatoes ( S. tuberosum cv. Ranger Russet) (Forsyth et al, 2016 ). Importantly, the molecular confirmation of transgenic lines showed a single copy of transgene in most of the regenerated events.…”

Genome Editing for Crop Improvement – Applications in Clonally Propagated Polyploids With a Focus on Potato (Solanum tuberosum L.)

“…Furthermore, studies related to technological advances in genome-editing in potato have been reported such as utilizing a native StU6 promoter to drive sgRNA expression, targeted insertion of transgenes, a Gateway system for rapid assembly of TALENs, and delivery of TALENs via agroinfiltration for rapid mutagenesis detection ( Wang et al, 2015 ; Forsyth et al, 2016 ; Kusano et al, 2016 ; Ma et al, 2017 ).…”