Gene Targeting Facilitated by Engineered Sequence-Specific Nucleases: Potential Applications for Crop Improvement

Abstract

Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world's population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining and are only rarely repaired via error-free homology-directed repair if an appropriate donor template is provided. Gene targeting (GT), i.e. the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

Keywords: Crop; Gene targeting; Sequence-specific nuclease.

Fig. 1

Various DSB repair pathways in higher plants. The primary repair mechanism for site-specific DSBs induced by SSNs is the error-prone NHEJ, which leads to random mutations. The error-free HDR creates precise knock-in or sequence substitutions when a specific repair template is provided.

Fig. 2

Possible working models of Agrobacterium transformation-mediated gene-targeting in plants. (A) T-DNA integration into the host plant genome. The single-stranded T-DNA region of the Ti plasmid forms a complex with VirD2, which along with other Vir proteins is transported to plant cells by the T4SS. VIP1, an Arabidopsis bZIP protein, together with Agrobacterium VirE2, forms a T-complex that is imported into the nucleus. In the nucleus, VirE2 and VIP are detached from the single-stranded T-DNA and VirD2 complex, and the T-DNA is integrated into the host plant genome. The SSNs, Cas9 and sgRNA are transcribed from the integrated T-DNA locus (or loci), although in some rare instances they might also be transcribed from nonintegrated T-DNA, such as extrachromosomal ds T-DNA (Gelvin 2017, Nishizawa-Yokoi and Toki 2021). (B) HDR-mediated GT pathways in plants. DSBs generated by SSNs are repaired via HDR. There are three possible repair templates: (i) randomly integrated T-DNA in a different locus, (ii) excised donor fragment from the integrated T-DNA locus and (iii) single-stranded T-DNA with VirD2.