Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants

Abstract

Gene targeting (GT) enables precise genome modification—e.g., the introduction of base substitutions—using donor DNA as a template. Combined with clean excision of the selection marker used to select GT cells, GT is expected to become a standard, generally applicable, base editing system. Previously, we demonstrated marker excision via a piggyBac transposon from GT-modified loci in rice. However, piggyBac-mediated marker excision has the limitation that it recognizes only the sequence TTAA. Recently, we proposed a novel and universal precise genome editing system consisting of GT with subsequent single-strand annealing (SSA)-mediated marker excision, which has, in principle, no limitation of target sequences. In this study, we introduced base substitutions into the microRNA miR172 target site of the OsCly1 gene—an ortholog of the barley Cleistogamy1 gene involved in cleistogamous flowering. To ensure efficient SSA, the GT vector harbors 1.2-kb overlapped sequences at both ends of a selection marker. The frequency of positive–negative selection-mediated GT using the vector with overlapped sequences was comparable with that achieved using vectors for piggyBac-mediated marker excision without overlapped sequences, with the frequency of SSA-mediated marker excision calculated as ~40% in the T0 generation. This frequency is thought to be adequate to produce marker-free cells, although it is lower than that achieved with piggyBac-mediated marker excision, which approaches 100%. To date, introduction of precise substitutions in discontinuous multiple bases of a targeted gene using base editors and the prime editing system based on CRISPR/Cas9 has been quite difficult. Here, using GT and our SSA-mediated marker excision system, we succeeded in the precise base substitution not only of single bases but also of artificial discontinuous multiple bases in the miR172 target site of the OsCly1 gene. Precise base substitution of miRNA target sites in target genes using this precise genome editing system will be a powerful tool in the production of valuable crops with improved traits.

Single‐strand annealing: Molecular mechanisms and potential applications in CRISPR‐Cas‐based precision genome editing

“…[ 83 ] Although natural SSA repair leads to sequence losses and chromosomal translocations or rearrangement, its mechanism could still be adopted to facilitate CRISPR/Cas‐based GT for removing selection markers. [ 21,22,84 ]…”

“…[ 90,91 ] However, the requirement of the TTAA motif at a predefined site for editing limits its application to some extent. [ 22,84 ]…”

Plant genome editing: ever more precise and wide reaching

“…SSA is one of the DNA DSB repair systems that use duplicated repeat sequences. This can be applied to any target sequence in principle, but the frequency of marker elimination by SSA is lower than that mediated by the piggyBac transposon (Endo et al, 2020; Ohtsuki et al, 2020). To date, reproducible reports of gene modification via positive–negative selection‐mediated GT are limited to rice.…”

Plant genome engineering from lab to field—a Keystone Symposia report

“…Similar to PiggyBac, this strategy leaves no footprint. Toki showed that this positive selection marker system can introduce mutations in the MIR172 binding site in rice 45 . Positive/negative selection–mediated gene targeting and subsequent removal of the positive selection marker using either the piggyBAC transposon or break‐induced SSA can be used to achieve any desired mutation without the need for sequence‐specific nucleases.…”