Generating broad-spectrum tolerance to ALS-inhibiting herbicides in rice by base editing

Abstract

Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control. We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase (ALS) gene to confer herbicide tolerance in rice. The four different missense mutations in the P171 codon, P171S, P171A, P171Y and P171F, exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors. For example, P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides. Interestingly, a novel triple mutant (P171F/G628E/G629S) had the highest tolerance to all five tested herbicides. Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron. Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties. base editing, herbicide tolerance, rice, acetolactate synthase (ALS)

The Development of Herbicide Resistance Crop Plants Using CRISPR/Cas9-Mediated Gene Editing

“…Fortunately, developing crop varieties harboring herbicide-resistant mutations that render crop tolerance to ALS-inhibiting herbicides by the CBE has been successfully applied in various crop species, including rice [61,95], maize [64], wheat [53,54], watermelon [55], oilseed rape [56], tobacco [57], tomato, and potato [58]. Similarly, crop tolerance to ACCaseinhibiting herbicides was also found in wheat via the CBE editor [53].…”

Genome editing: propelling the next generation of crop improvement

“…However, currently, there seems to be a shift to the CRISPR-Cas technique aiming for non-GMOs. Genome editing tools have already been applied for this purpose in some highly important crops, such as rice (LI et al, 2016;SUN et al, 2016;ZHANG et al, 2020a), soybean , maize (JIANG et al, 2020), oilseed rape (WU et al, 2020), tomato, and potato (VEILLET et al, 2019).…”

“…The mutation of ALS in specific amino acids results in resistance to herbicides such as sulfonylurea and imidazolinone. Herbicideresistant rice was obtained using the HR pathway, either by the introduction of multiple discrete point mutations in OsALS gene (SUN et al, 2016) or by prime editing, using a cytosine base editor to generate mutations in P171 and/or G628 codons of OsALS (ZHANG et al, 2020a). The prime editing strategy was also used to generate sulfonylurea herbicide-resistant maize (LI et al, 2020b), oilseed rape (WU et al, 2020), tomato, and potato (VEILLET et al, 2019).…”

“…For instance, in maize, CRISPR-Cas was used to delete an essential catalytic region of male sterility 26 (MS26) gene, translating a nonfunctional member of cytochrome P450 monooxygenase family and generating complete male-sterile plants (QI et al, 2020b). Similarly, disruption of TaNP1 genes, which encodes a putative glucose-methanol-choline oxidoreductase, resulted in complete male sterility in wheat (LI et al, 2020a). Multiplexdirected mutations driven by CRISPR-Cas are especially valuable in polyploid crops, which present redundant alleles, being very difficult to be generated/or gathered by other processes from traditional crop improvement.…”

Synthetic evolution of herbicide resistance using a T7 RNAP-based random DNA base editor

“…By contrast, for two-vector samples selected on 0.75 µM BS, we identified the enrichment of C>T causing the P93S substitution and G>C causing the G629R substitution (Figure 4b). Some of these ALS sites and the nearby sites G94, W548, and G629 [40][41][42][43] are known to confer resistance to BS. Interestingly, G629 appeared to be an important residue in our analysis, as substitutions at this site were enriched in samples transformed with the single-vector system selected on 0.5 µM BS and in samples transformed with the two-vector system on both concentration of BS (Figure 4b).…”