Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases

Abstract

Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species.

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

“…This approach allowed very efficient gene knock-in in tomato (3.65-11.66% of the transfected cells, which is about 10-fold higher than with conventional Agrobacterium delivery) using the replicon backbone of BeYDV [8]. Promising results were also obtained in potato using the same strategy [27]. 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).…”

“…Knock-in events presenting disrupted restriction sites will resist to the digestion and be available for a much more efficient and detectable amplification [11]. Furthermore, positive and/or negative selection markers, as well as a high selection pressure for gene modifications during the regeneration process (when the desired phenotype is amenable to it), can be used to increase the probability to see knock-in events [27][28][29]. For example, potato lines with edited ALS1 gene were easily detected on media containing the herbicide corresponding to the tolerance conferred by the knock-in of the modified allele [27].…”

“…Furthermore, positive and/or negative selection markers, as well as a high selection pressure for gene modifications during the regeneration process (when the desired phenotype is amenable to it), can be used to increase the probability to see knock-in events [27][28][29]. For example, potato lines with edited ALS1 gene were easily detected on media containing the herbicide corresponding to the tolerance conferred by the knock-in of the modified allele [27]. For transient transformation protocols on protoplasts, a preselection of the transfected cells can be done based on resistance genes inserted in the plasmid bearing the CRISPR system and the donor template.…”

“…Regarding the donor DNA, long donor templates (with long sequences of interest or long homology arms) can be delivered to the cells on the same or on another plasmid as the CRISPR-Cas system [2,6,8,27,41,69], or as linear dsDNA fragments, provided as free molecules [67] or released in planta from a plasmid by inserting them between CRISPR target sites [3,10,67]. Shorter templates (with short homology arms or small replacement sequences) can be introduced into the cells as ssDNA oligonucleotides (sense or antisense) [6,11,67,75].…”

“…Indeed, biolistic co-transformation in rice with plasmids bearing a donor cassette released in planta gave higher gene knock-in efficiency with additional free dsDNA donor fragments (36% of lines regenerated on selective media) than without (25% of lines regenerated on selective media) [67]. To strongly increase their copy number in the transformed cells, the sequences coding for the CRISPR-Cas9 system and the donor template can also be inserted into disarmed viral replicons of geminiviruses [8,27,89,90]. Other types of viral vectors, such as Tobacco Mosaic Virus or Tobacco Rattle Virus, have been long used for transient expression of recombinant proteins in plants [91], but these viral constructs are too low cargo for the delivery of the CRISPR coding elements all together.…”