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Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants

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Molecular Plant 2021年第2期14卷 352-360页

作者： Juan Li Rongfang Xu ruiying qin Xiaoshuang Liu Fanna Kong Pengcheng Wei Key Laboratory of Rice Genetic Breeding of Anhui Province Rice Research InstituteAnhui Academy of Agricultural SciencesHefei 230031P.R.China

Streptococcus pyogenes Cas9(SpCas9)is the most widely used genome editing tool in *** editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif(PAM),significantly limiting its scope of ***,five SpCas9 variants,SpCas9-NRRH,SpCas9-NRCH,SpCas9-NRTH,SpG,and SPRY,were developed to recognize non-canonical PAMs in human *** this study,these variants were engineered for plant genome editing,and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice(Oryza sativa)by stable ***,both cytosine base editors using a rat APOBEC1 or a human APO-BEC3a and adenine base editors using a directly evolved highly compatible TadA*-8e deaminase were developed from these SpCas9 *** results demonstrated that the developed SpCas9 variantsbased base editors readily generated conversions between C.G and T.A in the target sites with noncanonical PAMs in transgenic rice ***,the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.

关键词： SpCas9 variant PAM rice genome editing base editing

Increasing fidelity and efficiency by modifying cytidine base-editing systems in rice

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The Crop Journal 2020年第3期8卷 396-402页

作者： ruiying qin Shengxiang Liao Juan Li Hao Li Xiaoshuang Liu Jianbo Yang Pengcheng Wei Key Laboratory of Rice Genetics&Breeding of Anhui Province Institute of Rice ResearchAnhui Academy of Agricultural ScienceHefei 230031AnhuiChina

The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3(BE3) and targeted activation-induced cytidine deaminase(CDA)(target-AID) systems by coexpressing three copies of free uracil–DNA glycosylase(UDG) inhibitor(UGI). The editing efficiency of the improved BE3 and CDA systems reached as high as 88.9% and 85.7%, respectively, in regenerated rice plants, with a very low frequency of unwanted mutations. The low editing frequency of the BE3 system in the GC context could be overcome by the modified CDA system. These results provide a highfidelity and high-efficiency solution for rice genomic base editing.

关键词： CRISPR-Cas9 Base editing BE3 CDA Oryza sativa

Development of Plant Prime-Editing Systems for Precise Genome Editing

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Plant Communications 2020年第3期1卷 124-131页

作者： Rongfang Xu Juan Li Xiaoshuang Liu Tiaofeng Shan ruiying qin Pengcheng Wei Key Laboratory of Rice Genetics&Breeding Institute of Rice ResearchAnhui Academy of Agricultural ScienceHefei 230031China

Prime-editing systems have the capability to perform efficient and precise genome editing in human *** this study,we first developed a plant prime editor 2(pPE2)system and test its activity by generating a targeted mutation on an HPT^(-ATG) reporter in *** results showed that the pPE2 system could induce programmable editing at different genome *** transgenic T0 plants,pPE2-generated mutants occurred with 0%–31.3%frequency,suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse *** optimize editing efficiency,guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human ***,at the genomic sites tested in this study,pPE3 systems generated only comparable or even lower editing ***,we developed a surrogate pPE2 system by incorporating the HPT^(-ATG) reporter to enrich the prime-edited *** nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system,presumably due to the enhanced screening efficiency of edited *** together,our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.

关键词： prime editing CRISPR rice precise editing surrogate system

CRISPR/Cas9-Mediated Adenine Base Editing in Rice Genome

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Rice science 2019年第2期26卷 125-128,I0018-I0023页

作者： LI Hao qin ruiying LIU Xiaoshuang LIAO Shengxiang XU Rongfang YANG Jianbo WEI Pengcheng Key Laboratory of Rice Genetics & Breeding Institute of Rice ResearchAnhui Academy of Agricultural Science

Precise base editing is highly desired in plant functional genomic research and crop molecular breeding. In this study, we constructed a rice-codon optimized adenine base editor(ABE)-nC as9 tool that induced targeted A·T to G·C point mutation of a key single nucleotide polymorphism site in an important agricultural gene. Combined with the modified single-guide RNA variant, our plant ABE tool can efficiently achieve adenine base editing in the rice genome.

关键词： Precise optimized adenine agricultural gene

SDF5 Encoding P450 Protein Is Required for Internode Elongation in Rice

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Rice science 2021年第4期28卷 313-316,I0002-I0007页

作者： Yang Yachun Li Juan Li Hao Xu Zuntao qin ruiying Wu Wenge Wei Pengcheng Ding Yong YANG Jianbo Agricultural College Anhui Agricultural UniversityHefei 230036China Rice Research Institute Anhui Academy of Agricultural SciencesHefei 230031China School of Life Sciences University of Science and Technology of ChinaHefei 230026China

Plant height is a critical trait for yield in rice(Oryza sativa),and gibberellic acid(GA)is involved in modulating rice ***,we identified the semi-dwarf 5(sdf5)mutant by screening a rice T-DNA insertion mutant library using ***5 contains a T-DNA insertion that causes the ectopic expression of LOC_Os05g34325,which encodes a 14-α-demethylase cytochrome P450(CYP51)*** of the active GA species GA1 and GA4 were reduced in the sdf5 mutant,and the semi-dwarf phenotype of the sdf5 mutant was rescued by exogenous *** addition to its semi-dwarf height,the sdf5 mutant exhibited compact plant architecture,dark green leaves and low 1000-grain ***,these results suggest that SDF5 is involved in GA biosynthesis pathway and modulates rice height and plant development.

关键词： sativa dwarf Oryza

Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice

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Journal of Genetics and Genomics 2016年第8期43卷 529-532页

作者： Rongfang Xu Yachun Yang ruiying qin Hao Li Chunhong Qiu Li Li Pengcheng Wei Jianbo Yang Key Laboratory of Rice Genetic Breeding of Anhui Province Rice Research InstituteAnhui Academy of Agricultural SciencesHefei 230031China School of Agronomy Anhui Agricultural UniversityHefei 230036China

Most of the important agronomic traits in crop plants, such as yield, quality and stress response, are quantitative and jointly controlled by many genomic loci or major genes. Improving these complex traits depends on the combination of beneficial alleles at the quantitative trait loci （QTLs）. However, the conventional cross breeding method is extremely time-consuming and laborious for pyramiding multiple QTLs. In certain cases, this approach might be technically difficult because of close linkage between genes separately responsible for desirable and undesirable traits.

关键词： Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice

Developing a CRISPR/FrCas9 system for core promoter editing in rice

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aBIOTECH 2024年第2期5卷 189-195页

作者： Hui Wang Jian Ding Jingyan Zhu Xiaoshuang Liu Rongfang Xu ruiying qin Dongfang Gu Min Li Pengcheng Wei Juan Li College of Agronomy Anhui Agricultural UniversityHefei 230036China Key Laboratory of Rice Genetic Breeding of Anhui Province Rice Research InstituteAnhui Academy of Agricultural SciencesHefei 230031China Research Centre for Biological Breeding Technology Advance AcademyAnhui Agricultural UniversityHefei 230036China

Small mutations in the core promoter region of a gene may result in substantial changes in expression ***,targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible *** this study,we engineered a unique FrCas9 system derived from Faecalibaculum rodentium for plant genome *** findings indicate that this system is efficient in rice when the TATA sequence is used as a *** addition,FrCas9 demonstrated activity against all 16 possible NNTA PAMs,achieving an efficiency of up to 35.3%in calli and generating homozygous or biallelic mutations in 31.3%of the T_(0)transgenic plants.A proof-ofconcept experiment to examine editing of the rice WX core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose *** mutations and deletions were produced by bidirectional editing,mediated by FrCas9,using a single palindromic TATA sequence as a ***,we developed FrCas9-derived base editors capable of programmable conversion between AT and GC pairs in *** study highlights a versatile FrCas9 toolset for plant core promoter editing,offering great potential for the fine-tuning of gene expression and creating of new germplasms.

关键词： Genome editing FrCas9 CRISPR Core promoter Base editing

Prime editing-mediated precise knockin of protein tag sequences in the rice genome

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Plant Communications 2023年第3期4卷 14-17页

作者： Juan Li Jian Ding Jingyan Zhu Rongfang Xu Dongfang Gu Xiaoshuang Liu Jing Liang Chunhong Qiu Huanhuan Wang Min Li ruiying qin Pengcheng Wei Key Laboratory of Rice Genetic Breeding of Anhui Province Rice Research InstituteAnhui Academy of Agricultural SciencesHefei 230031China College of Agronomy Anhui Agricultural UniversityHefei 230036China These authors contributed equally to this article

Dear Editor,Accurately labeling proteins in living plant cells has long been a challenge and can be addressed by targeted insertion of tag sequences in a given *** optimized plant prime editors(PEs)enable efficient programmable installation of small insertions or deletions,including insertions of short sequences(Li et al.,2022a,2022b;Jiang et al.,2022;Xu et al.,2022;Zong et al.,2022;Zou et al.,2022).To investigate whether prime editing can be used to tag endogenous proteins in rice,we made use of the enpPE2 system described in our previous report(Li et al.,2022b).

关键词： insertion sequences precise

Development of plant cytosine base editors with the Cas12a system

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The Crop Journal 2023年第5期11卷 1451-1457页

作者： Huanhuan Wang Jing Liang Like Chen Bufang Deng Dongfang Gu Xiaoshuang Liu Shan Jin Rongfang Xu ruiying qin Yitong Zhu Liangxia Zhao Dourong Kou Yanjun Chen Yingli Jiang Juan Li Pengcheng Wei College of Agronomy Anhui Agricultural UniversityHefei 230036AnhuiChina Key Laboratory of Rice Genetic Breeding of Anhui Province Rice Research InstituteAnhui Academy of Agricultural SciencesHefei 230031AnhuiChina

Base editors of the Cas9 system have been widely used for precise nucleotide substitution in crops. In this study, Cas12a was applied to construct plant cytosine base editors(CBEs). The main elements of Cas12aCBEs were engineered and their efficiency was evaluated in stably transformed rice cells. An optimized ttCas12a-hyA3Bctd editor, consisting of a LbCas12a variant carrying catalytic inactive D832A and temperature-tolerance D156R double mutations, a truncated human APOBEC3B deaminase, a human RAD51 single-stranded DNA-binding domain, and double copies of UGI, outperformed other Cas12aCBEs in base editing efficiency. In T0transgenic rice plants, ttCas12a-hyA3Bctd edited an average of42.01% and a maximum of 68.75% of lines at six genomic targets. A-to-G conversions were generated in rice by an adenine base editor with a similar architecture to the optimized CBE. Our results provide preliminary evidence for the feasibility of robust and efficient plant Cas12a base editing systems, which could be useful for precise crop breeding.

关键词： Cas12a Base editing Rice ABE CBE