A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize

Abstract

ORCID IDs: 0000-0001-6179-5407 (Y.Zhou.); 0000-0001-7827-8211 (Q.-J.C.).The lack of efficient delivery methods is a major barrier to clustered regularly interspaced short palindromic repeats/CRISPRassociated protein (CRISPR/Cas)-mediated genome editing in many plant species. Combinations of morphogenic regulator (MR) genes and ternary vector systems are promising solutions to this problem. In this study, we first demonstrated that MR vectors greatly enhance maize (Zea mays) transformation. We then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, we developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. Our ternary vector system is composed of new pGreen-like binary vectors, here named pGreen3, and a pVS1-based virulence helper plasmid, which also functions as a replication helper for the pGreen3 vectors in Agrobacterium tumefaciens. The pGreen3 vectors were derived from the plasmid pRK2 and display advantages over pGreen2 vectors regarding both compatibility and stability. We demonstrated that the union of our ternary vector system with MR gene modules has additive effects in enhancing maize transformation and that this enhancement is especially evident in the transformation of recalcitrant maize inbred lines. Collectively, our ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.

Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize

“…Double peaks represent heterozygous or chimeric mutations, and an asterisk indicates a mutation induced by PE. The first mutation in #15 is the unwanted mutation introduced by the pegRNA scaffold vectors [10,11], pZ1PE3 and pZ1PE3b, for the same P165S mutation that are based on two different strategies, PE3 and PE3b [1], respectively (Fig. 1a, b).…”

“…We separately transformed the six PE vectors, pZ1PE3, pZ1PE3b, p4xZ1PE3-Csy4, p4xZ1PE3b-Csy4, pZ1WS-Csy4, and pZ1WS, into the engineered Agrobacterium strain LBA4404/pVS1-VIR2 to generate strains harboring the ternary vector system [11]. We used these strains separately to transform maize ND73, a B73-derived inbred line, according to published protocols [11].…”

Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize

“…We used the maize Ubi1 promoter to drive the expression of maize codon-optimized PE2 and the OsU3 and TaU3 promoters to drive the expression of pegRNA and sgRNA, respectively. In this way, we constructed two pGreen3 binary vectors [10,11], pZ1PE3 and pZ1PE3b, for the same P165S mutation that are based on two different strategies, PE3 and PE3b [1], respectively (Fig. 1a, b).…”

Agrobacterium-mediated delivery of CRISPR/Cas reagents for genome editing in plants enters an era of ternary vector systems

“…Agrobacterium-mediated genetic transformation (AMGT) is the preferred method of CRISPR/Cas reagent delivery; however, the process typically remains confined to limited species and genotypes, often non-elite cultivars with poor agronomic characteristics and low efficiencies of delivery (Altpeter et al, 2016;Lowe et al, 2016). Fortunately, the recent use of ternary vector systems coupled with morphogenic or developmental regulator (MR or DR) genes has greatly improved AMGT methods (Lowe et al, 2016;Anand et al, 2018;Che et al, 2018;Lowe et al, 2018;Zhang et al, 2019). In this article, we review the progress of AMGT-based delivery of CRISPR/Cas reagents and summarize recent developments from the perspective that Agrobacterium-mediated CRISPR/Cas genome editing in plants is entering an era of ternary vector systems, which are often integrated with MRs.…”