Efficient in planta gene targeting in Arabidopsis using egg cell‐specific expression of the Cas9 nuclease of Staphylococcus aureus

Abstract

Gene targeting (GT), the programmed change of genomic sequences by homologous recombination (HR), is still a major challenge in plants. We previously developed an in planta GT strategy by simultaneously releasing from the genome a dsDNA donor molecule and creating a double-stranded break (DSB) at a specific site within the targeted gene. Using Cas9 form Streptococcus pyogenes (SpCas9) under the control of a ubiquitin gene promoter, we obtained seeds harbouring GT events, although at a low frequency. In the present research we tested different developmentally controlled promotors and different kinds of DNA lesions for their ability to enhance GT of the acetolactate synthase (ALS) gene of Arabidopsis. For this purpose, we used Staphylococcus aureus Cas9 (SaCas9) nuclease and the SpCas9 nickase in various combinations. Thus, we analysed the effect of single-stranded break (SSB) activation of a targeted gene and/or the HR donor region. Moreover, we tested whether DSBs with 5' or 3' overhangs can improve in planta GT. Interestingly, the use of the SaCas9 nuclease controlled by an egg cell-specific promoter was the most efficient: depending on the line, in the very best case 6% of all seeds carried GT events. In a third of all lines, the targeting occurred around the 1% range of the tested seeds. Molecular analysis revealed that in about half of the cases perfect HR of both DSB ends occurred. Thus, using the improved technology, it should now be feasible to introduce any directed change into the Arabidopsis genome at will.

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

“…Attempts to boost in planta GT, by nCas‐mediated induction of SSBs instead of DSBs, did not result in comparable targeting efficiencies (Wolter et al ). Consequently, it is tempting to speculate whether the utilization of Cas12a, with its intrinsic property to induce short 5′ overhang DSBs, can improve the efficiency of in planta (GT), over the frequencies obtained with Cas9 (Wolter et al ). Due to its cleavage activity distal to its PAM, multiple rounds of DSB induction are possible after InDel formation, via NHEJ repair, as the crRNA is more tolerant to mismatches at this side of the target sequence.…”

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

“…Interestingly, for SpCas9 targeted mutagenesis frequencies could also be strongly enhanced via incubation at 37°C and this is applicable to different plant species [16]. Here, the repair template is stably integrated into the plant genome and GT can occur during the life cycle of the plant [21][22][23]. Gene targeting (GT), the induction of precise genome alterations by HR, is still highly challenging in plants as NHEJ is the preferred mechanism for DSB repair in somatic plant cells and most crops are still lacking efficient transformation and regeneration procedures.…”

“…A GT method independent on high transformation efficiencies is in planta GT. Here, the repair template is stably integrated into the plant genome and GT can occur during the life cycle of the plant [21][22][23]. In another approach, HR-based GT was enhanced by the use of geminivirus-based replicons carrying the homologous donor sequence.…”

Gene targeting in Arabidopsis via an all‐in‐one strategy that uses a translational enhancer to aid Cas9 expression

“…In addition, a previous all-in-one strategy was shown to successfully target the ALS locus in Arabidopsis. This strategy used a chimeric EC1.2/DD45 enhancer with the EC1.1 promoter to drive Cas9 expression in egg cells (Wolter et al, 2018). Therefore, we also generated an all-in-one construct that drives omega translational enhancer-Cas9 expression from a chimeric EC1.2/DD45 enhancer with the EC1.1 promoter (Figure 1a).…”

“…Another strategy that has been used to improve gene targeting efficiency in planta is to excise the donor fragment from the T-DNA by using sequence-specific nucleases (SSNs) (Wolter et al, 2018). To determine if excision of the HR donor improves the efficiency of our DD45-promoter-based all-in-one gene targeting system, we designed a construct in which the HR donor sequence was flanked by two recognition sites for the same sgRNA as endogenous ROS1 (Figure 1a).…”