Herbicide tolerance-assisted multiplex targeted nucleotide substitution in rice

Acetolactate synthase (ALS) catalyzes the initial step in the biosynthesis of branched-chain amino acids, and is highly conserved from bacteria to higher plants. ALS is encoded by a single copy gene in rice genome and is a target enzyme of several classes of herbicides. Although ALS mutations conferring herbicide-resistance property to plants are well documented, effect of Imazamox (IMZ) on rice and the mutations in ALS correlated with IMZ tolerance were unclear. In this article, the effect of IMZ on rice calli and seedlings in tissue culture conditions were evaluated. Also, the ALSA96V mutation was confirmed to improve IMZ tolerance of rice calli. Based on these results, ALS-assisted multiplex targeted base editing in rice was demonstrated in combination with Target-AID, a CRISPR/Cas9-cytidine deaminase fusion system [1], [2].


Value of the data
• Effective IMZ concentrations were determined to suppress rice callus proliferation and seedling growth in tissue culture conditions. • The data demonstrated that ALS A96V mutation confers IMZ tolerance to rice calli and is thus it is applicable as an endogenous selectable marker indicating the activity of Target-AID system. • Simultaneous engineering of multiplex traits of rice calli was successfully demonstrated by Target-AID in combination with ALS-assisted selection. This will contribute to more efficient selection of the prospective cells carrying desired mutations as IMZ tolerance provide a useful index of Target-AID activity.

Data
This article shows the optimization of an ALS-assisted screening strategy that facilitates more efficient targeted nucleotide substitutions in rice using Target-AID system. Optimal IMZ concentrations to inhibit rice callus proliferation and plant growth in tissue culture conditions were determined ( Fig. 1B and C). The conferring of IMZ tolerance to rice calli by introducing A96V mutation in ALS was confirmed (Table 1). On the basis of these results, simultaneous multiplex gene editing with ALSassisted Target-AID syrategy was demonstrated to introduce A96V mutation to endogenous ALS as well as restoration of EGFP (Fig. 2 B-D, Table 2).

Evaluation of Imazamox tolerance of rice in tissue culture conditions
Effects of IMZ concentration in N6D media [3] on rice callus were evaluated. N6D and N6DSE-IMZ medium containing IMZ at gradual concentrations (30, 50, 70 mg/L) were used in this assay. The proliferation of rice callus was strictly inhibited by 70 mg/L IMZ (Fig. 1B).
Wild type rice ALS gene was cloned from genomic DNA by PCR amplification using the appropriate primers numbered as 1 and 2 in Table 3. The ALS A96V gene was synthesized via overlapping PCR procedure using the primers 1-6. The DNA sequence of the clones carrying ALS genes were confirmed    using the primers 7-24. The cloned genes were installed to pRIT4, a derivative of binary vector pRIT3 [2] harboring a modified HPT gene [4]. The resultant vectors, pRIT4-ALS WT and pRIT4-ALS A96V (Fig. 1A) were introduced to rice calli by Agrobacterium-mediated transformation according to a previous report [5] using the plant media shown in Table 4. Transformed calli were selected on N6DSE-H40 medium over 3 weeks, then subcultured on N6DSE-H40IMZ70 medium to evaluate IMZ tolerance. After the selection over 2 months, 99.2% of calli introduced ALS A96V exhibited IMZ tolerance and proliferated on the media, whereas almost all the calli were sensitive to IMZ when introduced WT ALS ( Table 1). The minimum effective concentration of IMZ on rice seedlings in aseptic conditions were determined as follows. Wild-type rice seeds were germinated and grown on 1/2MS media containing IMZ at concentration of 0.01, 0.03, 0.05, 0.07, 0.09, 0.1 and 0.25 mg/L ( Table 4). The growth of the seedlings was analyzed by measuring their shoot length at 7 days after planting. As a result, seedling growth was remarkably suppressed by IMZ at 0.07 mg/L or higher concentration (Fig. 1C).

Multiplex editing of endogenous genes by Target-AID
To demonstrate the multiplex gene editing by Target-AID, the callus lines harboring pRIT3-mEGFP [2] were used in this experiment. Such calli were confirmed to carry dysfunctional EGFP (switch-mEGFP) containing a premature stop codon right after the initiation codon ( Fig. 2A). A vector for Target-AID system expressing nCas9(D10A)-PmCDA1 with gRNAs corresponding to endogenous ALS and mEGFP was introduced by Agrobacterium-mediated transformation. After selection on N6DSE-H40P50 media, the double transformants were subcultured on N6DSE-IMZ70 medium over 2 months. As a result, 3 callus lines exhibiting IMZ tolerance and EGFP expression were obtained from 124 double transformants (Table 2, Fig. 2B). The targeted nucleotide substitutions were confirmed by direct DNA sequencing analysis using primers 10, 24 for endogenous ALS and 25-27 for switch-mEGFP (Table 3). 2 calli were found to harbor both of the desired mutations leading to ALS A96V and functional recovery of EGFP gene ( Fig. 2A, C, D).