Targeted Deletion of the Wx Gene First Intron via CRISPR/Cas9 Signicantly Increases Grain Amylose Content in Rice With a Wxb Allele, but Not in Rice With a Wxa Allele

Background: Rice Waxy (Wx) gene plays a major role in seed amylose synthesis, and consequently controls grain amylose content. The expression of Wx gene is highly regulated at both transcriptional and post-transcriptional levels. Particularly, the GT/TT poplymorphism at the 5` splicing site of its 1 st intron greatly affects this intron’s splicing eciency and denes two predominant Wx alleles, Wx a and Wx b . Wx a rice often has intermediate to high amylose content, whereas Wx b rice has low to intermediate amylose content. A previous study indicates that rice Wx 1 st intron signicantly enhances gene expression when it is inserted into the 5` UTR (untranslated region) of a foreign gene. By deleting Wx 1 st intron with the CRISPR/Cas9 technology, we intended to create a totally noval Wx allele, and further to investigate how the intron removal affects Wx gene expression and rice grain amylose content. Results: CRISPR/Cas9-mediated targeted deletion of Wx 1 st intron was performed on 4 rice inbreds, KY131(Wx b ), X32(Wx b ), X35(Wx a ) and X55(Wx lv ). Complete deletion of the 1 st intron occurred in 8.6%-11.8% of the primary transformants of these 4 inbreds. Transgene-free, homozygous mutants were obtained. Their grain amylose content and Wx gene expression were analyzed. Compared to the amylose content of wild type plants, mutants’ amylose content was signicantly increased from 13.0% to about 24% in KY131 and X32 which both carried the Wx b allele. However, no signicant differenece in aylose content was observed between wild type plants and mutants of X35 and X55 which carried the Wx a and Wx lv allele, respectively. Results of Wx gene expression analysis on wild type plants and mutants showed a high consistence with their amylose content results. Mutants of KY131 and X32 accumulated much more steady mRNA transcripts than their wild type plants, while steady mRNA level remained somehow unchanged between wild type plants and mutants of X35 and X55. Grain quality including appearance quality and ECQ(eating and cooking quality) that are tightly linked to amylose content was also evalued on wild type plants and mutants, and data were presented and analyzed. Conclusions:This study presents a novel and fast strategy to increase amylose content for rice inbreds carrying a Wx b allele. Our data strongly suggest that rice Wx 1 st intron regulates Wx gene expression mainly at the post-transcriptinal level, not as previously thought that it inuences Wx gene transcription as well. In addition, removal of the rst intron creates a completely noval Wx allele. Further studies on this new Wx allele would provide invaluable insights into the regulation of Wx gene expression, which will help researchers to engineer more new alleles that leads to the breeding of rice cultivars with better eating and cooking quality. of their Wx genes was completely deleted. Further analyses indicated that these 14 mutants could be categorized into 3 mutant types according to their difference in the number of neucleotides deleted. These three mutant types were named as M1(-1041bp), M2(-1042bp), M3(-1043bp), repectively, with M2 as the predominant one (9/14) (Fig. 1D, Table 2). Alignment of the DNA sequence of these 3 mutant types with that of WT also revealed that, besides precise deletion of the entire rst intron (1021bp), extra 20-, 21- or 22bp were deleted in M1, M2 or M3 mutant, respectively (Fig. 1D). We predicted that the 5 s mRNA would respectively be 21- and 22bp shorter of wild type.


Background
Improving grain quality is one of the most important goals in rice (Oryza sativa L) breeding programs. Rice quality refers to the basic characteristics of rice in commodity circulation. It includes four main aspects, milling quality, appearance quality, cooking and eating quality and nutritional quality. Among them, appearance quality, cooking and taste quality (ECQ) are particularly important (Lau et al., 2015). ECQ is determined from amylose content (AC), gel consistency to the content of amylose, rice was divided into four types, glutinous (AC < 2%), soft, intermediate to low and high (AC > 25%) (Pandey et al., 2012). Generally, the higher amylose content in rice grain is, the less sticky and harder the cooked rice is, resulting in poor taste (Jobling, 2004;Juliano, 1992). However, rice with too low amylose content is too sticky and soft. Thus, it is more popular with intermediate to low amylose content in rice.
The Waxy (Wx) gene encodes granule-bound starch synthase I(GBSSI), which was cloned by Wang et al in 1990 (Sano, 1984;Sano et al., 1985;Wang et al., 1990). The Wx gene is a major gene controlling amylose content in rice endosperm,and plays a decisive role on rice ECQ (Tian et al., 2009). At present, there are great differences in amylose content among cultivated rice varieties. Such vast differences mainly come fromWx gene allele variation. At least 9 Wx natural alleles have been discovered and identi ed in rice. Those alleles include Wx a , Wx b , Wx in , Wx op /Wx hp , Wx mq , Wx mp , Wx lv , Wx la /Wx mw , and wx ( homozygous edited mutants free of transgenic components could be selected through progeny separation .Compared with the traditional transgenic methods, the CRISPR/Cas9 technology is more prone to the changes of natural mutations and has more potential to be applied in production practice. Recently, CRISPR/Cas9 mediated rice Wx gene editing have been intensevly reported. These reporters can be grouped into 4 categories. First, researchers have created new glutinous rice by completely knocking out the Wx gene Zhang et al., 2018b). Second,by editing the key cisacting elements on the Wx gene promoter, researchers have achieved ne-tune of AC in the Wx b background (Huang et al., 2020a;Zeng et al., 2020). Third, researchers have tried to regulate rice AC by manipulating the splicing e ciency of Wx gene at the post-transcriptional level (Zeng et al., 2020). Fourth, researchers have used CRISPR/Cas9-mediated base editor to is modulate rice AC by modifying GBSSI enzyme activity(Xu et al., 2020). These studies have not only paved new avenues for rice AC improvement, but also generated a plenty of new Wx alleles and added precious new knowledge about the regulation of rice Wx gene expression and the modulation of GBSSI enzyme activity. In addition, a previous study shows that the rst intron of the rice Wx gene greatly enhances foreign gene expression in rice protoplasts, but not in tobacco protoplasts. When it is inserted into the 5` UTR region between the CaMV 35S promoter and GUS (β-glucoronidase ) coding sequence, GUS expression is increased 15-fold in rice protoplasts (Li et al., 1995), strongly suggesting that the rst intron of the Wx gene might play an important role in regulating the Wx gene expression, presumably at the transcriptional level.
In the study, we exploited the CRISPR/Cas9 gene editing technology to remove the entire rst intron of the Wx gene. The intron removal created a totally noval Wx allele, and signi cantly increased the amylose content in rice inbreds with the Wx b allele, while the amylose content was not changed much in rice inbreds with the Wx a allele.

Results
CRISPR/Cas9-mediated deletion of Wx 1st intron To investigate how Wx 1st intron regulates amylose content of rice grains, we decided to remove the entire 1st intron using CRISPR/Cas9 tenchnology. We chose four rice inbreds with different genetic background as our testing materials. Kongyu131 (KY131) was an elite japonica inbred and carried a typic Wx b allele. The other three X32, X35 and X55 were indica inbreds from our breeders, carrying Wx b , Wx a or Wx lv allele, respectively. The typical Wx lv allele of X55 carried the same GT polymorphism as Wx a allele at the well-de ned GT/TT polymorphism site that differentiates Wx a and Wx b alleles . We designed two target sites, Target1 and Target2 which was located at the 5 of Wx 1st intron, respectively (Fig. 1A). An edit vector (Fig. 1B) expressing CRISPR/Cas9, two gRNAs (guide RNA, gRNA1 for target1 and gRNA2 for target2), and CP4/EPSPS (as selection marker) was delivered into rice cells through Agrobacterium-mediated transformation. A total of 314 glyphosate-resistant transgenic plants were generated for these 4 rice inbreds (Table 1). Our PCR assays on all the 314 primary transformants detected 33 mutants with one or two copies of the rst intron removed, deletion e ciency ranging from 8.6-11.85% (Fig. 1C, Table 1).These 33 intron deleted mutants included 12 for KY131, 8 for X32,5 for X35 and 8 forX55 (Fig. 1C, Table 1). We further investigated if off-target occurred in our experiments. Uisng the online sfoftware CRISPR-P2.0(http://crispr.hzau.edu.cn/CRISPR2/), we identi ed two potential off-target sites for both gRNAs used in this study. PCR ampli cation and DNA sequencing of predicted off-target sites were performed on both T0 mutants and partial of their T1 offsprings. As shown in Supplementary Table S1, no off-target was detected at the putative off-target loci in both tested T0 plants and their T1 offsprings.
Identi cation of transgene-free, homozygous mutants T1 seeds of the primary transformants with normal seed setting rate and other agronomic traits were selected for further screening of transgene-free, homozygous mutants. As shown in Tables 1, 4, 4, 4 and 2 target mutant lines were botained for KY131, X32, X55 and X35, respectively. Sequencing these 14 lines con rmed that the rst intron of their Wx genes was completely deleted. Further analyses indicated that these 14 mutants could be categorized into 3 mutant types according to their difference in the number of neucleotides deleted. These three mutant types were named as M1(-1041bp), M2(-1042bp), M3(-1043bp), repectively, with M2 as the predominant one (9/14) (Fig. 1D, Table 2). Alignment of the DNA sequence of these 3 mutant types with that of WT also revealed that, besides precise deletion of the entire rst intron (1021bp), extra 20-, 21-or 22bp were deleted in M1, M2 or M3 mutant, respectively (Fig. 1D). We predicted that the 5 s mRNA would respectively be 20-, 21-and 22bp shorter than that of wild type. a n d 3  1D). AC values reported are mean ± SD. Letters following the AC mean values stands for the levels of difference, a, not signi cantly different, b, signi cantly different (P < 0.01, t-test).

Wx gene expression was signi cantly increased in KY131 and X32 mutants
To investigate the expression of Wx gene in intron-deleted mutants, the relative amount of Wx gene mRNA in 10 DAP (days after pollination) seeds of different mutant plants and WT plants was detected by qRT-PCR. Results were shown in Fig. 2. Compared to KY131 and X32 WT plants that both carried Wx b allele, the intron-deleted mutants of KY131 and X32 plants accumulated about 1-2 times more stable mRNA ( Fig. 2A), suggesting that removal of the rst intron signi cantly affects Wx gene expression in a Wx b background. However, the relative expression leves of Wx gene between X35 and X55 WT plants ( both have the Wx a allele ) and their intron-deleted mutants did not show much difference ( Fig. 2A), demonstrating that removal of the rst intron does not affect the Wx gene expression in a Wx a background.
As aforementioned that the 5 s mRNA would respectively be 20, 21 or 22bp shorter than that of WT (Fig. 1D), we wanted to know whether these differences would in uence Wx gene expression. Our Wx gene expression results and AC data demonstrated that there was not much change among two X35 mutants, X35-CR-1(M3),-CR-2 M2 , and WT X35 ( Table 2, Fig. 1A), suggesting that mutant type M2 and M3 had similar expression level as the WT. Similar notion coud be derived from comparison between four X55 mutants (all M2 mutant type) and WT X55 in terms of their Wx expression and AC (Table 2, Fig. 1A). Thus, we concluded that the extra 20-22bp deletion in the 5` UTR of intron deleted mutants had not much in uence on Wx gene expression.
Deletion of Wx 1st intron substantially increased grain amylose content of KY131 and X32 mutants The grain amylose content of transgene-free, homozygous mutants ( rst generation) and together with their corresponding WT plants was measured, and the results were shown in Table 2. The amylose content of KY131 and X32 mutants signi cantly increased from 13% to about 24%, while the amylose content of X35 and X55 mutants had no signi cant difference ( Table 2, Fig. 2B,). These amylose content results were well correlated to our Wx gene expression data. Signi cantly increased relative expression of Wx gene was only observed in KY131 and X32 mutants that showed signi cant AC increase, but not in X35 and X55 mutants that showed similar AC as their corresponding WT plants ( Fig. 2A). We further analyzed the grain amylose content of second generation KY131 and X32 mutants. Results showed that the increased level of amylose content was consistent in both generations (Fig. 3A,3B), suggesting that the amylose content change in mutants were genetically stable. Grain quality evaluation and physicochemical property analysis of mutant grains It is well established that grain amylose content is closely linked to grain quality and affects grain physicochemical properties such as GC and GT ( X32. Compared with grains of the WT plants, grains of 4 mutant plants showed no signi cant difference in gelatinization temperature/ASV (alkakli spreading value) (Table 3). However, their amylose content increase did slightly decrease the Gel consisitency of rice grains (  Fig. 3B). In addition, milling quality was also not much different between the grains of KY131 and X32 WT plants and their mutants ( Table 3). Grain transparency evaluation demonstrated that polished rice grains of X32 intron-deleted mutants had better transparency than that of X32 WT plants, indicating appearance quality was improved in X32 intron-deleted mutants (Table 3, Fig. 4A, 4B). However, the improvement of appearance quality observed in X32 mutants was not found in KY131 intron-deleted mutants (Table 3, Fig. 4C, 4D). We further inspected whether amylose content change in uences the structure of starch granules by scanning electron microscopy (SEM) of transverse mature endosperm sections. Our results revealed no obivios difference in the morphology of starch granules from the endosperm between mutants and wild types ( Fig. 4E-P). Notes: Values reported are mean ± SEM. Letters following the mean values stands for the levels of difference, a, not signi cantly different, b, signi cantly different (P < 0.01, t-test). AC, amylose content, GC, gel consistency, ASV, alkali spreading value; PV, peak viscosity; HPV, through viscosity or hot paste viscosity; CPV, nal viscosity or cool paste viscosity; BDV, breakdown viscosity (BDV = PV-HPV); SBV, setback viscosity (SBV = CPV-PV); CSV, consistency viscosity (CSV = CPV-HPV); PeT, peak time; PaT, pasting temperature. All the viscosity parameters were expressed in rapid visco units (RVU).

Conclusions
The amylose content (AC) in rice endosperm is an important factor affecting the rice eating and cooking quality (ECQ) (Tian et al., 2009). However, vast differences in regional commsumer preference, market demend and functionality makes ECQ hard to de ne and standardize. In general, South Asians favor long, slender rice with a high AC and hard gel consistency (GC), while Southeast Asians have a preference for long grains with intermediate AC and soft GC. Such differences often hinder the spread of elite varieties to other countries whose rice markets demand rice with a different AC. Developing e cient, low-cost technology to modulate grain AC will provide a reliable solution to overcome this barrier. In addition, the development of specialized rice consumption creates more diversi ed demands for rice AC. For example, changing market demands more and better quality rice specialized for rice wine industry and for patients with diabetes or high blood pressure in recent years (Sun et al., 2017).Therefore, it is of great commercial value to develop biotechnological methods to accurately adjust rice grain AC to satisfy diverse commsumers.
With the rapid advance of CRISPR/Cas-mediated gene editing technologies (Gao, 2021) for rice AC improvement, shed new lights on the understanding of the molecular regulation of Wx gene expression and the modulation of GBSSI enzyme activity, and importantly generated an array of noval Wx alleles that have valuable application potential. In this study, we developed a new strategy to modify rice AC. We deleted the entire rst intron of the rice Wx gene in both Wx a and Wx b backgrounds using the CRISPR/Cas9 technology. Removal of the rst intron from inbreds KY131 and X32 with a Wx b allele signi cantly increased grain AC, by more than 10%, from 13% to about 24.0% (Table 2 Fig . 2). However, such a phenomena was not observed with inbreds X35 and X55 carrying a Wx a allele. Grain AC of WT X35, X55 and their intron-deleted mutants remained about the same ( Table 2, Fig. 2). Our method will provide an e cient and rapid way to convert intermediate AC rice cultivars (mostly with a Wx b allele) to high AC cultivars, which facilitates elite rice cultivars of intermediate AC adapted to new plantation regions and comsumer markets that favor high AC cultivars.
Deletion of the entire rst intron generated a complete noval Wx allele which has other implications. First, removal of the rst intron allowed us to gain insights into its regulation on Wx gene expression. Previous studies have showed that the GT/TT SNP at the 5' splicing junction of the rst intron is a major posttranscriptional regulation factor to affect rice grain AC (Cai et al., 1984;et al., 1998;Samadder et al., 2008). By eliminating the splicing process of the rst intron, we demonstrated that rice seeds accumulated about 1-2 times more amount of stable mRNA and their amylose content increased by about 10% (from 13% to about 24%) after the rst intron removal under Wx b background ( Table 2, Fig. 2), while stable mRNA accumulation and seed AC remained about the same after the rst intron deleted under the Wx a background ( Table 2, Fig. 2). This gives us a quantitative view on how much the G to T SNP posttranscriptionally affects the expression of the Wx gene. A previous study also demonstrates that the rst intron of the rice Wx gene stimulates the expression of a foreign gene in rice protoplasts when it is placed in the 5' UTR region between CaMV 35S promoter and GUS coding sequence, indicating that the rst intron could act as a transcriptional enhancer (Li et al., 1995). This notion migh have solid ground given the fact that quite a few rst introns with big size (~ 1.0kb or more) and located within the 5` UTR of highly expressed genes such as rice Actin1, maize Ubi1, indeed function as transcriptional stimulators (Rose, 2008(Rose, , 2019. However, our data demonstrated that rice seeds carrying the Wx a gene with or without the rst intron accumulated about the same amount of stable mRNA and produced about the same amount of amylose ( Table 2 Engineering new Wx alleles that perform better under high temperature will be invaluable to rice production, particularly as global warming increasingly poses threats on the world food security.
In conclusion, our study presented a noval and e cient strategy to modify rice grain amylose content, particularly, to generate high amylose content rice from a rice carrying a Wx b allele. By deleting the entire rst intron, we created a completely noval Wx allele. Futher analyses on the rst intron deleted mutants provided new insights into the regulation of Wx gene expression. And the new Wx allele could serve as an excellent material for further engineering new Wx alleles that perform better under high temperature and improve rice eating and cooking quality.

Construction of the CRISPR/Cas9 vector and plant transformation
The CRISPR/Cas9 vector targeting the rst intron of the Wx gene was constructed as previously described (Zhang et al., 2014b). The vector used in this study was based on the vector pCambia1300 backbone. The editing vector pZZT477 contained a Cas9 expression cassette driven by sugarcane Ubi4 promoter, a CP4 -EPSPS gene cassette (as selection marker) driven by CaMV 35S promoter, Two sgRNA expression cassettes respectively driven by rice U3 or U6 snRNA promoters (Fig. 1). The editing vector pZZT477 was transferred into Agrobacterium tumefaciens strain EHA105 by electroporation and consequently delivered into KY131, X32, X35 and X55 cells via Agrobacterium-mediated transformation as previously described (Hiei et al., 1997).

RNA Extraction and RT-PCR analysis
Total RNA was extracted from grains after 10 days of grain-lling using the TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche). Quantitative RT-PCR analysed were conducted to amplify Wx gene on an Applied Biosystems 7900HT instrument with 2× SYBR Green PCR Master Mix (Applied Biosystems).

Scanning Electron Microscopy of Starch Granules
Rice grains were dried in an oven at 42°C for 2 days and cooled in a desiccator. Cross sections of the samples were manually snapped and sputter-coated with gold palladium on copper studs. Magni cations of 500× and 2000× were used to observe endosperm and starch granule morphology.

Evaluation of Rice Quality
The rice quality was measured following the procedure described in GB/T 15683 − 2008 and NY/T 83-2017, and each sample was tested for 3 times. RVA pasting properties were detected with a Rapid Visco Analyzer (RVA) according to the manufacture's instruction (NewPort Sci. Co., Australia).

Statistical Analysis
The data were analyzed by using Student's unpaired t-test in the Microsoft Excel. Differences were considered to be signi cant at P < 0.05 or P < 0.01.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. SupplementaryTables.docx