Fine‐tuning the amylose content of rice by precise base editing of the Wx gene DOI Creative Commons
Yang Xu, Qiupeng Lin, Xiufeng Li

и другие.

Plant Biotechnology Journal, Год журнала: 2020, Номер 19(1), С. 11 - 13

Опубликована: Июнь 17, 2020

The genetic diversity and phenotypic variability of crop agronomic traits is valued by breeders for their benefits in breeding but are limited most target traits. Genome editing has proved to be a powerful tool quick efficient creation continuous beneficial variation (Eshed Lippman, 2019). rice Waxy (Wx) gene (LOC_Os06g04200) encodes granule-bound starch synthase I (GBSSI), which determines the amylose content (AC) endosperm controlling synthesis. This one major contributors eating cooking quality (ECQ) (Li et al., 2016), an attribute that receiving increased attention society because improvement people's living standards. Rice AC ranges from 0 ~30% depending on presence different Wx alleles, with Wxa(relatively high more than 20%) Wxb (intermediate 14 ~18%) being alleles found indica japonica varieties, respectively (Teng 2012). Amino acid changes Wx/GBSSI protein can affect grain, as well-known 'soft rice' varieties (AC 7%–10%) genotypes Wxop/hp, WxmqorWxmp (Zhu 2015), all have non-synonymous mutations N-terminal domain (Momma Fujimoto, As moderately low (<12%), become popular commercially Gilbert, 2018), both traditional molecular approaches including CRISPR/Cas9-mediated knockout (Ma 2015; Zhang 2018) been used mutate reduce grain. However, only number mutants generated, far fewer needed meet diverse demands ECQ. We hypothesized grain could fine-turned generating series novel amino substitution(s) close allele responsible sites (such residues 158th inWxmq or Wxmp, 191th 165th Wxop/hp allele) state-of-the-art base editing. Based requirements cytidine editors (CBEs) (Zong 2017), we designed three sgRNAs targeting third (target site1, TS1), fourth site 2, TS2) fifth site3, TS3) exons (Figure 1a), were mentioned sites. cloned into vector pH-nCas9-PBE generate vectors PBE-TS1, PBE-TS2 PBE-TS3, respectively. resulting plasmids individually introduced cultivar Nipponbare (NIP) Agrobacterium-mediated transformation. A total 5, 10 7 independent T0 transgenic lines, respectively, 5 2 representative edited lines 1b) taken T1 generation; T-DNA-free homozygous individuals then chosen analysed detail. observed variety mutation types position substitutions within window; these reflected present parental suggesting T0alleles faithfully transmitted next generation 1b). Using TS1, line, Wxm5 (from line B7-2/6), carrying C2, 3, 5-to-T2, transition led P124F R125W was obtained; using TS2, four Wxm6 B6-29, G6, 7-to-A6, leading G159K mutation), Wxm7 B2-25, G6-to-C6 transversion G159A Wxm8 G1-to-A1 transversion, D161N mutations) Wxm9 B1-68, G4-to-T4 G6-to-A6 transition, G159E V160F identified; TS3, two Wxm10 B2-21, C5, 6-to-T5, 6 T178I mutation) Wxm11 C5-to-G5 C6-to-T6 T178S obtained 1c). In addition, seven (Wxm5-Wxm11), failed find any potential off-target 1d). To determine effect AC, measured apparent contents (AACs) grains mutant NIP (Wxb) control Nangeng9108 (NG9108) (Wxmp) 1e). Notably, had AAC (1.4 ± 0.2%) glutinous rice. AACs (11.9 0.1%), (11.3 (9.8 (7.9 0.1%) significantly lower (14.4 0.2%), comparable NG9108 (9.6 0.2%). (5.8 (4.2 lay between those Wxm5. GBSSI activities developing seeds Wx-edited days after flowering ranged 231.5 16.5 712.1 54.1 nmol/g/min 1f), NIP. reduced likely due amount 1g). These results demonstrate TS1-TS3 indeed abundance activity decrease seeds. general, appearance milled (especially transparency grain) negatively correlated 2018). 7%–10% (e.g. NG9108) semi-translucent while < 2% opaque. compared (10% moisture) (T2 generation) NG9108. indicated Figure 1h, opaque rice-like, consistent AAC. Wxm8, like Interestingly, Wxm6, andWxm10, 9.8%–11.9%, tended rather NG9108, almost transparent semi-translucent, indicating successfully generated germ plasms (~10%) without affecting achieved confirmed other Jingeng818 (JG818) Suijing18 (SJ18), NIP, exampleWxm5, 1i), strategy this study reliable fine-tune elite varieties. summary, base-editing system create 1.4%–11.9% goal over range 0%–12% enrich materials available breeders. Furthermore, speculated C-terminal domain) and/or Wxa) further extend AC. shows it possible obtain substituting many individual acids critical domains genes economically important provides new breeding. work supported grants National Transgenic Science Technology Program (2019ZX08010-003), Natural Foundation China (31701511), Jiangsu Province (BK20170610), Key R&D (2018YFA0900600) State Laboratory Plant Cell Chromosome Engineering (PCCE-KF-2020-01). authors submitted patent application based reported paper. J. Y., C. G. Y. X. research; X., Q. L., F. W., Z. C., W. F., T., J., R. performed B. contributed writing; Q-H. wrote manuscript.

Язык: Английский

Precise fine-turning of GhTFL1 by base editing tools defines ideal cotton plant architecture DOI Creative Commons
Guanying Wang, Fuqiu Wang, Zhongping Xu

и другие.

Genome biology, Год журнала: 2024, Номер 25(1)

Опубликована: Фев. 26, 2024

CRISPR/Cas-derived base editor enables precise editing of target sites and has been widely used for basic research crop genetic improvement. However, the efficiency editors at different targets varies greatly.

Язык: Английский

Процитировано

20
Antagonizing cis- regulatory elements of a conserved flowering gene mediate developmental robustness DOI Creative Commons
Amy Lanctot, Anat Hendelman,

Peter Udilovich

и другие.

Proceedings of the National Academy of Sciences, Год журнала: 2025, Номер 122(8)

Опубликована: Фев. 18, 2025

Developmental transitions require precise temporal and spatial control of gene expression. In plants, such regulation is critical for flower formation, which involves the progressive maturation stem cell populations within shoot meristems to floral meristems, followed by rapid sequential differentiation into organs. Across plant taxa, these are orchestrated F-box transcriptional cofactor UNUSUAL FLORAL ORGANS ( UFO ). The conserved pleiotropic functions offer a useful framework investigating how evolutionary processes have shaped intricate cis- key developmental genes. By pinpointing promoter sequence in an accessible chromatin region tomato ortholog , we engineered vivo series regulatory alleles that caused both loss- gain-of-function defects. These mutant phenotypes were linked disruptions predicted transcription factor binding sites known activators repressors. Allelic combinations revealed dosage-dependent interactions between opposing alleles, influencing penetrance expressivity phenotypes. phenotypic differences support robustness development requires expression dosage. Bridging our analysis Arabidopsis found although homologous sequences dispersed promoter, they maintain similar over development. However, from disrupting differ due differing patterns . Our study underscores complex dynamic genes demonstrates short stretches recruit activating repressing machinery robustness.

Язык: Английский

Процитировано

2
Solanum pan-genetics reveals paralogues as contingencies in crop engineering DOI Creative Commons
Matthias Benoit, Katharine M. Jenike, James W. Satterlee

и другие.

Nature, Год журнала: 2025, Номер unknown

Опубликована: Март 5, 2025

Язык: Английский

Процитировано

2
Fine-tuning sugar content in strawberry DOI Creative Commons

Sinian Xing,

Kunling Chen,

Haocheng Zhu

и другие.

Genome biology, Год журнала: 2020, Номер 21(1)

Опубликована: Сен. 3, 2020

Abstract Fine-tuning quantitative traits for continuous subtle phenotypes is highly advantageous. We engineer the conserved upstream open reading frame (uORF) of FvebZIPs1.1 in strawberry ( Fragaria vesca ), using base editor A3A-PBE. Seven novel alleles are generated. Sugar content homozygous T1 mutant lines 33.9–83.6% higher than that wild-type. also recover a series transgene-free mutants with 35 genotypes containing continuum sugar content. All could be immediately fixed subsequent generations by asexual reproduction. Genome editing coupled reproduction offers tremendous opportunities trait improvement.

Язык: Английский

Процитировано

129
Fine‐tuning the amylose content of rice by precise base editing of the Wx gene DOI Creative Commons
Yang Xu, Qiupeng Lin, Xiufeng Li

и другие.

Plant Biotechnology Journal, Год журнала: 2020, Номер 19(1), С. 11 - 13

Опубликована: Июнь 17, 2020

The genetic diversity and phenotypic variability of crop agronomic traits is valued by breeders for their benefits in breeding but are limited most target traits. Genome editing has proved to be a powerful tool quick efficient creation continuous beneficial variation (Eshed Lippman, 2019). rice Waxy (Wx) gene (LOC_Os06g04200) encodes granule-bound starch synthase I (GBSSI), which determines the amylose content (AC) endosperm controlling synthesis. This one major contributors eating cooking quality (ECQ) (Li et al., 2016), an attribute that receiving increased attention society because improvement people's living standards. Rice AC ranges from 0 ~30% depending on presence different Wx alleles, with Wxa(relatively high more than 20%) Wxb (intermediate 14 ~18%) being alleles found indica japonica varieties, respectively (Teng 2012). Amino acid changes Wx/GBSSI protein can affect grain, as well-known 'soft rice' varieties (AC 7%–10%) genotypes Wxop/hp, WxmqorWxmp (Zhu 2015), all have non-synonymous mutations N-terminal domain (Momma Fujimoto, As moderately low (<12%), become popular commercially Gilbert, 2018), both traditional molecular approaches including CRISPR/Cas9-mediated knockout (Ma 2015; Zhang 2018) been used mutate reduce grain. However, only number mutants generated, far fewer needed meet diverse demands ECQ. We hypothesized grain could fine-turned generating series novel amino substitution(s) close allele responsible sites (such residues 158th inWxmq or Wxmp, 191th 165th Wxop/hp allele) state-of-the-art base editing. Based requirements cytidine editors (CBEs) (Zong 2017), we designed three sgRNAs targeting third (target site1, TS1), fourth site 2, TS2) fifth site3, TS3) exons (Figure 1a), were mentioned sites. cloned into vector pH-nCas9-PBE generate vectors PBE-TS1, PBE-TS2 PBE-TS3, respectively. resulting plasmids individually introduced cultivar Nipponbare (NIP) Agrobacterium-mediated transformation. A total 5, 10 7 independent T0 transgenic lines, respectively, 5 2 representative edited lines 1b) taken T1 generation; T-DNA-free homozygous individuals then chosen analysed detail. observed variety mutation types position substitutions within window; these reflected present parental suggesting T0alleles faithfully transmitted next generation 1b). Using TS1, line, Wxm5 (from line B7-2/6), carrying C2, 3, 5-to-T2, transition led P124F R125W was obtained; using TS2, four Wxm6 B6-29, G6, 7-to-A6, leading G159K mutation), Wxm7 B2-25, G6-to-C6 transversion G159A Wxm8 G1-to-A1 transversion, D161N mutations) Wxm9 B1-68, G4-to-T4 G6-to-A6 transition, G159E V160F identified; TS3, two Wxm10 B2-21, C5, 6-to-T5, 6 T178I mutation) Wxm11 C5-to-G5 C6-to-T6 T178S obtained 1c). In addition, seven (Wxm5-Wxm11), failed find any potential off-target 1d). To determine effect AC, measured apparent contents (AACs) grains mutant NIP (Wxb) control Nangeng9108 (NG9108) (Wxmp) 1e). Notably, had AAC (1.4 ± 0.2%) glutinous rice. AACs (11.9 0.1%), (11.3 (9.8 (7.9 0.1%) significantly lower (14.4 0.2%), comparable NG9108 (9.6 0.2%). (5.8 (4.2 lay between those Wxm5. GBSSI activities developing seeds Wx-edited days after flowering ranged 231.5 16.5 712.1 54.1 nmol/g/min 1f), NIP. reduced likely due amount 1g). These results demonstrate TS1-TS3 indeed abundance activity decrease seeds. general, appearance milled (especially transparency grain) negatively correlated 2018). 7%–10% (e.g. NG9108) semi-translucent while < 2% opaque. compared (10% moisture) (T2 generation) NG9108. indicated Figure 1h, opaque rice-like, consistent AAC. Wxm8, like Interestingly, Wxm6, andWxm10, 9.8%–11.9%, tended rather NG9108, almost transparent semi-translucent, indicating successfully generated germ plasms (~10%) without affecting achieved confirmed other Jingeng818 (JG818) Suijing18 (SJ18), NIP, exampleWxm5, 1i), strategy this study reliable fine-tune elite varieties. summary, base-editing system create 1.4%–11.9% goal over range 0%–12% enrich materials available breeders. Furthermore, speculated C-terminal domain) and/or Wxa) further extend AC. shows it possible obtain substituting many individual acids critical domains genes economically important provides new breeding. work supported grants National Transgenic Science Technology Program (2019ZX08010-003), Natural Foundation China (31701511), Jiangsu Province (BK20170610), Key R&D (2018YFA0900600) State Laboratory Plant Cell Chromosome Engineering (PCCE-KF-2020-01). authors submitted patent application based reported paper. J. Y., C. G. Y. X. research; X., Q. L., F. W., Z. C., W. F., T., J., R. performed B. contributed writing; Q-H. wrote manuscript.

Язык: Английский

Процитировано

123