CRISPR‐S: an active interference element for a rapid and inexpensive selection of genome‐edited, transgene‐free rice plants

The CRISPR/Cas9-based genome editing tool has been used in diverse applications related to plant research, including for crop improvement (Sun et al., 2016; Liu et al., 2017). Mutant plants may be generated via transient transformations or DNA-free editing (Liang et al., 2017). However, plant genomes are often edited during the production of transgenic plants, in a process that involves the identification of targeted edits in regenerated T0 plants and the subsequent elimination of transgenes in T1 plants (Sun et al., 2016). 
 
This article is protected by copyright. All rights reserved.

IN vector to form a hairpin structure, which was then introduced into a pCAMBIA-1300 vector between the double 35S (d35S) promoter and the Nos terminator (nos). We inserted the d35S-hpRNAi-nos element into the HpaI site near the left border of a CRISPR/Cas9 vector, pHun4c12, to construct a new CRISPR/Cas9 vector, pHun4c12s ( Figure 1a). To test the utility of pHun4c12s, we constructed another CRISPR/Cas9 expression vector, pHun4c12s-lct1, to target OsLCT1, which encodes a Cd transporter (Uraguchi et al., 2011). Ninety-six independent transgenic T 0 plants were generated via Agrobacterium tumefaciens-mediated transformation of japonica rice genotype Xidao #1 cells with pHun4c12s-lct1 and analysed further.
First, we tested the susceptibility of the T 0 plants to bentazon using a foliar spray. Tillers of T 0 plants were separated and grown as two subplants. One normally growing subplant of each T 0 plant was treated with a bentazon solution (2000 mg/L) by spraying until droplets were visible on leaves. About 1 week later, 29 T 0 subplants appeared to be susceptible and eventually died (T 0 -S plants; Figure 1b). The other 67 T 0 plants were not visibly affected (T 0 -R plants).
Second, we sequenced the OsLCT1 target region of the T 0 plants. All 29 T 0 -S plants were affected by homozygous (aa), biallelic (aa 0 ) or heterozygous (Aa) mutations in the target region. Mutations were not detected in the remaining plants. Thus, bentazon susceptibility was 100% correlated with the targeted mutations in T 0 plants.
Third, we analysed the abundance of Cas9 and CYP81A6 transcripts in T 0 -R and T 0 -S plants using a quantitative real-time PCR (qRT-PCR). Overall, the Cas9 transcript was significantly more abundant in T 0 -S plants than in T 0 -R plants (Figure 1c). In contrast, the abundance of the CYP81A6 transcript was lowest in the T 0 -S plants (Figure 1d), suggesting that CYP81A6-hpRNAi was more highly expressed in these plants. The expression of CYP81A6-hpRNAi resulted in the degradation of CYP81A6 transcripts via RNAi in transgene-expressing, T 0 -S plants. In contrast, the transgene-silenced T 0 plants remained resistant to bentazon ( Figure 1e). These observations imply that CYP81A6-hpRNAi enables the efficient selection of genome-edited T 0 plants.
Our system was also designed to simplify the selection of transgenefree T 1 plants. We grew 72 seedlings for each of 16 T 1 lines derived from homozygous or biallelic OsLCT1 mutant T 0 plants treated them with 1000 mg/L bentazon in a foliar spray (approximately 100 mL/m 2 ) at around the four-leaf stage. About 4 days later, we observed that all Xidao #1 seedlings were growing normally, but some of the T 1 seedlings started dehydrating and eventually died (Figure 1f). Furthermore, we proved that all bentazon-susceptible plants were transgenic, while all bentazon-resistant plants lacked T-DNA. The segregation of T 1 plants derived from a homozygous or biallelic genome-edited T 0 plant is presented in Figure 1e, in which the genome-edited, transgene-free plants are resistant to bentazon because the #2 #3 #4 #5 #7 #9 #11 #13 #18 #19 #20 #22 #23 #26 #27 #29 Transgenic plants produced by A. tumefaciens-mediated transformation often carry one or two copies of T-DNA (Collier et al., 2017). The segregation ratio observed for bentazon susceptibility was consistent with this fact in all T 1 lines except for Line #19 (Figure 1g). All Line #19 T 1 seedlings were susceptible to bentazon and died. Based on the 3:1 segregation ratio of its T 1 population (Figure 1g), the Line #2 T 0 plant was expected to have a single T-DNA insertion and was therefore used as the control for the qRT-PCR analysis. The Line #19 T 0 plant and its T 1 progenies appeared to have the same T-DNA copy number, and they all had double the number of insertions of the Line #2 T 0 plant (Figure 1h). This implies that T-DNA copies were incorporated into sister chromosomes in Line #19 T 0 plant. Otherwise, variability in the T-DNA copy numbers among the T 1 plants would have been detected.
To test the utility of CRISPR-S for other genes, we constructed a pHun4c12s-frg vector to target OsBADH2 (Bradbury et al., 2005). We transformed nine rice genotypes and obtained 4-22 T 0 plants for each genotype. All T 0 plants susceptible to bentazon were confirmed to carry mutations. Similarly, transgene-free, OsBADH2-edited T 1 plants were identified following a bentazon treatment of T 1 seedlings.
The results of our proof-of-concept study revealed that the Cas9 expression level could be indirectly estimated using a marker trait generated by an RNAi element incorporated into the CRISPR/Cas9 expression vector. This new CRISPR/Cas9 system provides a relatively simple way of identifying and eliminating T 0 plants in which the genome has not been edited. More importantly, our method enables the selection of transgene-free T 1 plants, with almost no cost, confirming the value of this system.
Antibiotic or herbicide resistance genes in binary vectors are important for selecting transgenic plants. However, they cannot be directly used to select transgene-free T 1 plants because they would kill the plants. Seed-localized fluorescent reporters have been used to discriminate between transgenic and nontransgenic seeds in a few plant species. Unfortunately, they are unsuitable for species that produce seeds with hulls or glumes such as rice. Although our system has been demonstrated in rice, it may be possible to develop similar systems in other plant species. Highly conserved homologs of CYP81A6 have been detected in monocots. Thus, the bentazon-susceptibility trait may also be used in these plant species. Furthermore, other suitable marker traits can be generated by down-regulating the expression of other genes to introduce visible morphological changes to leaf shape and colour, among other characteristics.