Improved Eating and Cooking Quality of indica Rice Cultivar YK17 via Adenine Base Editing of Wxa Allele of Granule-Bound Starch Synthase I (GBSS I)

Mahmuda Binte Monsur, Cao Ni, Wei Xiangjin, Xie Lihong, Jiao Guiai, Tang Shaoqing, Nese Sreenivasulu, Shao Gaoneng, Hu Peisong

Letter

Improved Eating and Cooking Quality ofRice Cultivar YK17 via Adenine Base Editing ofWxAllele of Granule-Bound Starch Synthase I (GBSS I)

Mahmuda Binte Monsur1, #, Cao Ni1, #, Wei Xiangjin1, Xie Lihong1, Jiao Guiai1, Tang Shaoqing1, Nese Sreenivasulu2, Shao Gaoneng1, Hu Peisong1

(State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China; Applied Functional Genomics Cluster, Grain Quality and Nutrition Centre, Strategic Innovation Platform, International Rice Research Institute, Los Banos 4030, the Philippines; These authors contributed equally to this work)

Amylose content (AC) is the key determinant of eating and cooking quality (ECQ) of rice. The majorWxallele of granule-bound starch synthase I (GBSS I) inrice produces higher AC, making rice hard and dry after cooking. Recent work has improved ECQ ofrice via clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) or cytosine base editing (CBE) techniques. However, base editing has not yet been applied to modify theWxallele ofrice. We utilized a novel precise adenine base editing (ABE) tool to generate three mutants ofcultivar Zhongjiazao 17 (YK17) with reduced AC while other ECQ parameters, such as gel consistency (GC) and alkali spreading value, were maintained. Our study demonstrated improvement of ECQ ofrice and will help rice breeders satisfy consumers.

AC, which determines ECQ in rice, is mainly controlled by(), a gene encoding GBSS I (Teng et al, 2012; Zhang et al, 2021). Variation at thelocus is largely responsible for the diversity of AC levels (Tian et al, 2009; Biselli et al, 2014; Zhang et al, 2019). Alleles includingWx,Wx,Wx,Wx,Wx,Wx,Wx,Wxandhave been reported (Cai et al, 1998; Sato et al, 2002; Larkin and Park, 2003; Wanchana et al, 2003; Mikami et al, 2008; Liu et al, 2009; Yang et al, 2013; Li et al, 2020; Zhang et al, 2021). Higher AC and lower GC values correlate with poor taste due to hard texture, whereas moderate AC rice (15%–20%) with higher GC values (60–80 mm) leading to soft texture is preferred by most consumers worldwide (Zeng et al, 2020). Invarieties, alleleWxgives rise to 25%–30% AC (Wang et al, 1995), while invarieties alleleWxproduces 15%–18% AC (Zhang et al, 2018). The ‘Old’ natural and ‘New’ editedalleles in rice crops will be helpful in developing novel rice varieties and for further quality improvement (Huang et al, 2020a).

Traditional successive backcrossing methods have been used to improve ECQ by introgressingWx, with moderate AC, intorice. However, traditional breeding strategies are always time-consuming and difficult to break close linkages with unfavourable traits. Recently, CRISPR/Cas9-based genomic editing techniques have been widely used for editing thegene of rice. The glutinous rice was generated by CRISPR/ Cas9-targeted mutagenesis of thegene in elite rice varieties (Zhang et al, 2018; Fei et al, 2019). Moreover, novelalleles with fine-tuned amylose levels can be created and rice grain quality can be improved by promoter editing using CRISPR/Cas9 (Huang et al, 2020b). Zeng et al (2020) reported thequantitative regulation ofexpression by CRISPR/Cas9-based promoter and 5-UTR-intron editing, improving grain quality in rice. Recently, base editing is an advanced CRISPR-based tool that ensures base conversion in a target gene (Komor et al, 2016; Gaudelli et al, 2017; Molla and Yang, 2019; Monsur et al, 2020). CBE, which converts cytosine (C) to thymine (T), has been applied successfully to fine-tune AC inrice (Xu et al, 2020). Modifying theWxallele using base editing strategies has the potential to generaterice with desirable AC to weak the textural preferences.

In this study, we focused on improving ECQ ofrice cultivars by decreasing AC via a precise ABE tool that can convert adenine (A) to guanine (G) (Li et al, 2018). We selectedrice cultivar YK17 as the super rice cultivar of China with high seed-setting rate and yield, early maturation, moderate plant morphology and good disease resistance (Barman et al, 2019). However, it produces high AC (27.3%) owning to theWxallele (Fig. S1). In our work, 10 target sites were selected randomly in the conserveddomain with glycosyl transferase activity supported by CRISPRdirect (http://crispr.dbcls.jp/) and CRISPR-GE (http://skl.scau.edu.cn/). The sgRNAs were modified into enhanced sgRNA (esgRNA) to make an optimal form for plant ABE-7 (PABE-7). Using the adenosine deaminase, nuclear localization sequences PABE-7 and esgRNA, Li et al (2018) created the efficient vector pH- PABE-7-esgRNA. The constructed plasmids were individually transformed into YK17 by the- mediated method. Independent transgenic plants were generated for each transformant and all the target sequences were sequenced. Target sites on exons 2, 6 and 8 ofWxhad mutations (Fig. 1-A and Table S1). Mutation efficiency varied from 37.50% to 69.23% in T0generation (Table S2). Sequencing results showed that homozygous mutants in three target sites, hereafter named as transgenicto(,and), were obtained in T0generation (Fig. S2), which were further used to generate the T1plants. T-DNA segregation confirmation of T0and T1plants was conducted throughselection on media (Fig. S3). Only T-DNA free plants were used for further studies (Fig. 1-B to -E). Inand, base conversion of A to G resulted in asparagine (AC) conversion to aspartate (AC), whereas in, glutamine (CG) converted to arginine (CG) (Fig. 1-A). Also, the positions of varied nucleotide (1246, 1634 and 496) and amino acid (247, 306 and 128) in consistent with three mutants are showed in Fig. 1-A. Multiple sequence alignment reflected that the mutated amino acid inwas less conserved in comparison to those inand(Fig. S4). Furthermore, five putative off-target sites were screened for each target site and no off-target effects were detected in all the T-DNA-free plants by the DNA sequencing method (Table S3).

Brown rice from wild type YK17 had a chalkiness appearance, as didand, whereashad opaque phenotypic appearance (Fig. 1-F to -I). For confirmation, we measured the AC and found(25.6%),(16.7%) and(3.4%) had lower AC than that of wild type YK17 (27.3%) (Fig. 1-J), which was consistent with the conclusion that the varied amino acids is less conserved incompared toand. Generally, based on AC values, rice grain is classified into five groups: waxy (0%–5%), very low (5%– 12%), low (12%–20%), intermediate (20%–25%) and high (25%–33%) groups (Juliano, 1998; Zhang et al, 2018). Notably,had low AC, whereashad AC as low as glutinous rice (Fig. 1-J) and fell into the waxy category. GC values decreased slightly in(76 mm) and(77 mm) and increased in(90 mm) compared to YK17 (81 mm) (Fig. 1-K). Thus, GC values forandare in the desirable range (60–80 mm). Alkali spreading value, which stands for gelatinization temperature (GT), did not differ significantly between the wild type and mutants (Fig. 1-L). Those results indicated that the amino acid substitutions in Waxy led to ECQ alterations. In addition, we used rapid visco analysis to evaluate grain starch quality. A decreasing trend of viscosity was observed with decreasing AC (Fig. 1-M). Further, we checked total GBSS I via western blot. Compared to YK17, the mutations inandled to increased GBSS I accumulation in 10-day filling grains (Fig. 1-N), especially in, probably caused by the reduction of Waxy protein degradation. Agronomic traits like grain length, grain width and grain thickness were also measured, and most phenotypes increased in the mutants compared to wild type. However, 1000-grain weight increased only in(Table S4).

Fig. 1. Desirable amylose content ofrice cultivar YK17 via adenine base editing ofgene.

A, Structure of Wxand the mutations in edited T1lines. Protospacer-adjacent motifs (PAMs) include three bases (NGG) with a redunderline. The red letters indicate altered bases. Q, Glutamine; R,Arginine; N, Asparagine; D, Aspartate. B–E, Gross morphologies of the wild type YK17 and its mutants,and. Scale bars are 10 cm. F–I, Appearance and transverse sections of brown rice of YK17 and mutants. Scale bars are 1 mm. J, Amylose content. K, Gel consistency. L, Alkali spreading values of the wild type and mutants. Data are Mean ± SD (= 3) in J–L. Samples with different lowercase letters show a significant difference at< 0.05 according to the Duncan’s test. M, Pasting properties of endosperm starch of YK17 and mutants.N, Western blotting of GBSS I in YK17 and mutant rice grains at 10 d after flowering. Actin was used as an internal control.NIP, Nipponbare; YK17, Zhongjiazao 17.

In conclusion, using an ABE tool, we created novel allelic variations withingene mutants having different AC (3.4%, 16.7% and 25.6%) with varied ECQ properties. Notably, we achieved better ECQ inwith desirable target of moderate AC (16.7%) and higher GC (77 mm), together with the improvement of viscosity, which prevents retrogradation. This study demonstrated a significant strategy for the improvement of AC inriceby introducing novel alleles through genome editing techniques, where most cultivars carry theWxallele and exhibit high AC.

ACKNOWLEDGEMENTs

This study was supported by the China National Key Research and Development Program (Grant No. 2020YFE0202300), the Central Public-Interest Scientific Institution Basal Research Fund of China (Grant Nos. Y2020PT07and Y2020YJ09), and the International Science & Technology Innovation Program of Chinese Academy of Agricultural Sciences, China (Grant No. CAAS-ZDRW202109). We are thankful to Prof. Gao Caixia for providing the ABE vector pH-PABE-7-esgRNA.

SUPPLEMENTAl DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

File S1. Methods.

Fig. S1.genotype of YK17.

Fig. S2. Sequencing results of target loci of YK17 and three mutants (,and).

Fig. S3. Selection of-resistant transgenic plants.

Fig. S4. Alignment of amino acids ofWxmutants,andconserved in different plant species.

Table S1. Primers used in this study.

Table S2. Mutation efficiency in T0generation.

Table S3. Identification of off-target effects.

Table S4. Agronomic traits of YK17 and mutants (,and).

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Hu Peisong (hupeisong@caas.cn); Shao Gaoneng (shaogaoneng@caas.cn)

11 October 2020;

4 March 2021

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http://dx.doi.org/10.1016/j.rsci.2021.07.003