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Science China(Life Sciences) 2020年第10期63卷 1491-1498页

作者： Yu Zhang qiang Zhang qi-jun chen State Key Laboratory of Plant Physiology and Biochemistry College of Biological SciencesChina Agricultural UniversityBeijing 100193China Center for Crop Functional Genomics and Molecular Breeding China Agricultural UniversityBeijing 100193China

Lack of appropriate methods for delivery of genome-editing reagents is a major barrier to CRISPR/Cas-mediated genome editing in ***-mediated genetic transformation(AMGT)is the preferred method of CRISPR/Cas reagent delivery,and researchers have recently made great improvements to this *** this article,we review the development of AMGT and AMGT-based delivery of CRISPR/Cas *** give an overview of the development of AMGT vectors including binary vector,superbinary vector,dual binary vector,and ternary vector *** also review the progress in Agrobacterium genomics and Agrobacterium genetic engineering for optimal *** focus in particular on the ternary vector system and the resources we *** summary,it is our opinion that Agrobacterium-mediated CRISPR/Cas genome editing in plants is entering an era of ternary vector systems,which are often integrated with morphogenic *** new vectors described in this article are available from Addgene and/or MolecularCloud for sharing with academic investigators for noncommercial research.

关键词： genome editing CRISPR/Cas ternary vector system delivery Agrobacterium-mediated transformation

Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice

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Plant Communications 2024年第2期5卷 67-78页

作者： Ajay Gupta Bo Liu Saad Raza qi-jun chen Bing Yang Division of Plant Science and Technology Bond Life Sciences CenterUniversity of MissouriColumbiaMO 65211USA State Key Laboratory of Plant Physiology and Biochemistry College of Biological SciencesChina Agricultural UniversityBeijing 100193China Center for Crop Functional Genomics and Molecular Breeding China Agricultural UniversityBeijing 100193China Donald Danforth Plant Science Center St.LouisMO 63132USA

Prime editing(PE)technology enables precise alterations in the genetic code of a genome of *** offers great potential for identifying major agronomically important genes in plants and editing them into superior variants,ideally targeting multiple loci simultaneously to realize the collective effects of the ***,we report the development of a modular assembly-based multiplex PE system in rice and demon-strate its efficacy in editing up to four genes in a single transformation *** duplex PE(DPE)system achieved a co-editing efficiency of 46.1%in the T0 generation,converting TFIIAg5 to xa5 and xa23 to *** resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae(Xoo)strains in the T1 *** addition,we success-fully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele,achieving a co-editing rate of 57.14%.Furthermore,with the quadruple PE(QPE)system,we edited four genes—two for herbicide tolerance(OsEPSPS1 and OsALS1)and two for Xoo resistance(TFIIAg5 and OsSWEET11a)—using one construct,with a co-editing efficiency of 43.5%for all four genes in the T0 *** performed multiplex PE usingfive more constructs,including two for triplex PE(TPE)and three for QPE,each targeting a different set of *** editing rates were dependent on the activity of pegRNA and/or *** instance,optimization of ngRNA increased the PE rates for one of the targets(OsSPL13)from 0%to 30%but did not improve editing at another target(OsGS2).Overall,our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents,making it feasible for more labs to utilize PE for their editing *** have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.

关键词： prime editing rice bacterial blight herbicide tolerance multiplex genome editing

Enhanced editing efficiency in Arabidopsis with a LbCas12a variant harboring D156R and E795L mutations

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aBIOTECH 2024年第2期5卷 117-126页

作者： Cuiping Xin Dexin qiao junya Wang Wei Sun Zhenghong Cao Yu Lu Yuanyuan Jiang Yiping Chai Xue-chen Wang qi-jun chen State Key Laboratory of Plant Environmental Resilience College of Biological SciencesChina Agricultural UniversityBeijing 100193People’s Republic of China

Cas12a(Cpf1),a Class 2 Type V CRISPR/Cas nuclease,has several unique attributes for genome editing and may provide a valuable alternative to ***,a low editing efficiency due to temperature sensitivity and insufficient cleavage activity of the Cas12a nuclease are major obstacles to its broad *** this report,we generated two variants,ttAsCas12 Ultra and ttLbCas12a Ultra harboring three(E174R,M537R,and F870L)or two(D156R and E795L)mutations,respectively,by combining the mutations from the temperature-tolerant variants ttAsCas12a(E174R)and ttLbCas12a(D156R),and those from the highly active variants AsCas12a Ultra(M537R and F870L)and LbCas12a Ultra(E795L).We compared editing efficiencies of the five resulting Cas12a variants(LbCas12a,ttLbCas12a,ttLbCas12a Ultra,AsCas12a Ultra,and ttAsCas12 Ultra)at six target sites of four genes in Arabidopsis(Arabidopsis thaliana).The variant ttLbCas12a Ultra,harboring the D156R and E795L mutations,exhibited the highest editing efficiency of all variants tested in Arabidopsis and can be used to generate homozygous or biallelic mutants in a single generation in Arabidopsis plants grown at 22 *** addition,optimization of ttLbCas12a Ultra,by varying nuclear localization signal sequences and codon usage,further greatly improved editing ***,our results indicate that ttLbCas12a Ultra is a valuable alternative to Cas9 for editing genes or promoters in Arabidopsis.

关键词： Genome editing CRISPR/Cas Cas12a ttLbCas12a ultra Arabidopsis

Analysis of var genes cloned from a Plasmodium falcivarum isolate in China

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Asian Pacific Journal of Tropical Medicine 2012年第2期5卷 85-90页

作者： Ning Jiang Li Meng Hui-jun Lu Wei Kang Shuai Peng Wei-qing Pan Ji-Gang Yin qi-jun chen Key Laboratory of Zoonosis Ministry of EducationInstitute of ZoonosisJilin University Department of Pathogen Biology Laboratory of Parasitology Institute of Pathogen BiologyChinese Academy of Medical Sciences

Objective:To analyse the rar gene repertoire and characterise the rhondroitin sulphate A (CSA)-binding activity of the Duffy-binding like(I)BI.) domains encoded by the var2csa gene of a Plasmodium falciparum(***) isolate in Hainan Province,***:The sequences of var DBL1 regions were PCR-amplified,sequenced and the sequence characteristics was bioinformalically *** proteins encoded by the var2csa genes were expressed and *** binding activities of the recombinant proteins to CSA receptor was detected by ELISA ***:Fifty six unique DBI.a sequences were obtained,and the sequences represented similar diversity to the var genes of the genome parasite *** are two var2csa genes in the *** isolated from Hainan *** in other falciparum parasites such as HB3,the two var2csa genes are more *** receptor-binding capacity of DBL-5εand DBI.-6 e domains of HN var2CSA was ***:This work represented the diversity of rar genes of a *** isolate in China.

关键词： Malaria Plasmodium falciparum Antigenic variation Var gene

Optimized prime editing efficiently generates heritable mutations in maize

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Journal of Integrative Plant Biology 2023年第4期65卷 900-906页

作者： Dexin qiao junya Wang Min‐Hui Lu Cuiping Xin Yiping Chai Yuanyuan Jiang Wei Sun Zhenghong Cao Siyi Guo Xue‐chen Wang qi‐jun chen State Key Laboratory of Plant Physiology and Biochemistry College of Biological SciencesChina Agricultural UniversityBeijing 100193China Center for Crop Functional Genomics and Molecular Breeding China Agricultural UniversityBeijing 100193China Collaborative Innovation Center of Crop Stress Biology Henan ProvinceInstitute of Plant Stress BiologySchool of Life ScienceHenan UniversityKaifeng 475004China

Low efficiency is the main obstacle to using prime editing in maize(Zea mays).Recently,prime-editing efficiency was greatly improved in mammalian cells and rice(Oryza sativa)plants by engineering primeediting guide RNAs(pegRNAs),optimizing the prime editor(PE)protein,and manipulating cellular determinants of prime *** this study,we tested PEs optimized via these three strategies in *** demonstrated that the ePE5max system,composed of PEmax,epegRNAs(pegRNA-evopreQ.1),nicking single guide RNAs(sgRNAs),and MLH1dn,efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS),acetolactate synthase(ALS),or acetyl CoA carboxylase(ACCase)***,we demonstrate that the ePE5max system has sufficient efficiency to generate heritable(homozygous or heterozygous)mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.

关键词： CRISPR/Cas epegRNAs herbicide resistance maize MLH1dn prime editing

PE6c greatly enhances prime editing in transgenic rice plants

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Journal of Integrative Plant Biology 2024年第9期66卷 1864-1870页

作者： Zhenghong Cao Wei Sun Dexin qiao junya Wang Siyun Li Xiaohan Liu Cuiping Xin Yu Lu Syeda Leeda Gul Xue‐chen Wang qi-jun chen State Key Laboratory of Plant Environmental Resilience College of Biological SciencesChina Agricultural UniversityBeijing100193China Center for Crop Functional Genomics and Molecular Breeding China Agricultural UniversityBeijing 100193China

Prime editing is a versatile CRISPR/Cas-based precise genome-editing technique for crop *** new types of prime editors(PEs)named PE6a–d were recently generated using evolved and engineered reverse transcriptase(RT)variants from three different *** this study,we tested the editing efficiencies of four PE6 variants and two additional PE6 constructs with double-RT modules in transgenic rice(Oryza sativa)***6c,with an evolved and engineered RT variant from the yeast Tf1 retrotransposon,yielded the highest prime-editing *** average fold change in the editing efficiency of PE6c compared with PEmax exceeded 3.5 across 18 agronomically important target sites from 15 *** also demonstrated the feasibility of using two RT modules to improve prime-editing *** results suggest that PE6c or its derivatives would be an excellent choice for prime editing in monocot *** addition,our findings have laid a foundation for prime-editing-based breeding of rice varieties with enhanced agronomically important traits.

关键词： genome editing PE6 PE6c prime editing prime editor rice

Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS

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Molecular Plant 2022年第11期15卷 1646-1649页

作者： Yuanyuan Jiang Yiping Chai Dexin qiao junya Wang Cuiping Xin Wei Sun Zhenghong Cao Yu Zhang Yun Zhou Xue-chen Wang qi-jun chen State Key Laboratory of Plant Physiology and Biochemistry College of Biological SciencesChina Agricultural UniversityBeijing 100193China Collaborative Innovation Center of Crop Stress Biology KaifengHenan Province 475004China Institute of Plant Stress Biology School of Life ScienceHenan UniversityKaifeng 475004China Center for Crop Functional Genomics and Molecular Breeding China Agricultural UniversityBeijing 100193China

Dear Editor,Prime editing is a novel and universal CRISPR-Cas-derived precise genome-editing technology and has great potentials for applications in basic plant research and crop molecular breeding(Anzalone et al.,2019).Although low efficiency has restrained the original prime editors(PEs)from being used as a routine tool for precise genome editing in plants,an iterative update of the PEs is removing this obstacle(Lin et al.,2021;Xu et al.,2022).Recently,the Liu group reported three optimization strategies for improving prime editing efficiency(chen et al.,2021;Nelson et al.,2022).The first strategy is based on engineered prime editing guide RNAs(epegRNAs),which were generated by incorporating structured RNA motifs to the 3′terminus of *** strategy enhances pegRNA stability and prevents degradation of the 3′extension(Nelson et al.,2022).The second strategy is based on the optimized PE2 protein(PEmax),which harbors a SpCas9 variant with increased nuclease activity,an additional nuclear localization signal(NLS)sequences,and a new linker between nCas9 and reverse transcriptase(chen et al.,2021).The third strategy is based on inhibition of DNA mismatch repair(MMR)in cells(chen et al.,2021).In this work,we tested the optimized PEs generated with these three strategies in rice,demonstrating that the optimized PEs greatly improved prime editing efficiency in *** named the two optimized PEs ePE3max and ePE5max:the former is comprised of the PEmax protein,an epegRNA with evopreQ1 appended to its 3′end,and a nicking sgRNA;the latter is comprised of the ePE3max system and a dominant negative OsMLH1 variant for inhibiting *** the two optimized PEs,we efficiently generated homozygous and heterozygous T173I,A174V,and P177S(TAP-IVS)mutation in EPSPS in rice,which lays a solid foundation for rice non-transgenic glyphosate-resistance breeding.

关键词： breeding precise editing

Modulatory effect of International standard Scalp Acupuncture on brain activation in the elderly as revealed by resting-state fMRI

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Neural Regeneration Research 2019年第12期14卷 2126-2131页

作者： Wai-Yeung Chung Song-Yan Liu Jing-Chun Gao Yi-Jing Jiang Jing Zhang Shan-Shan Qu Ji-Ping Zhang Xiao-Long Tan jun-qi chen Sheng-Xu Wang School of Traditional Chinese Medicine Southern Medical University Guangzhou Guangdong Province China School of Chinese Medicine The Chinese University of Hong Kong Shatin Hong Kong Special Administrative Region China Department of Neurology China-Japan Union Hospital of Jilin University Changchun Jilin Province China Department of Rehabilitation Medicine Third Affiliated Hospital of Southern Medical University Guangzhou Guangdong Province China Department of Rehabilitation Medicine Rehabilitation Hospital Fujian University of Traditional Chinese Medicine Fuzhou Fujian Province China Department of Medical Image Third Affiliated Hospital of Southern Medical University Guangzhou Guangdong Province China

The specific mechanisms by which acupuncture affects the central nervous system are unclear. In the International Standard Scalp Acupuncture system, acupuncture needles are applied at the middle line of the vertex, anterior parietal-temporal oblique line, and the posterior parietal-temporal oblique line. We conducted a single-arm prospective clinical trial in which seven healthy elderly volunteers (three men and four women;50–70 years old) received International Standard Scalp Acupuncture at MS5 (the mid-sagittal line between Baihui (DU20) and qianding (DU21)), the left MS6 (line joining Sishencong (EX-HN1) and Xuanli (GB6)), and the left MS7 (line joining DU20 and Qubin (GB7)). After acupuncture, resting-state functional magnetic resonance imaging demonstrated changes in the fractional amplitude of low frequency fluctuations and regional homogeneity in various areas, showing remarkable enhancement of regional homogeneity in the bilateral anterior cingulate, left medial frontal gyrus, supramarginal gyrus, right middle frontal gyrus, and inferior frontal gyrus. Functional connectivity based on a seed region at the right middle frontal gyrus (42, 51, 9) decreased at the bilateral medial superior frontal gyrus. Our data preliminarily indicates that the international standard scalp acupuncture in healthy elderly participants specifcally enhances the correlation between the brain regions involved in cognition and implementation of the brain network regulation system and the surrounding adjacent brain regions. The study was approved by the Ethics Committee of the China-Japan Union Hospital at Jilin University, China, on July 18, 2016 (approval No. 2016ks043).

关键词： nerve regeneration resting-state functional magnetic resonance imaging International Standard Scalp Acupuncture acupoint specificity brain functional connectivity healthy elderly volunteers low frequency fluctuation regional homogeneity functional connectivity neural regeneration

Long-term acupuncture treatment has a multitargeting regulation on multiple brain regions in rats with Alzheimer＇s disease：a positron emission tomography study

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Neural Regeneration Research 2017年第7期12卷 1159-1165页

作者： Yang-jia Lu Xiao-wen Cai Gui-feng Zhang Yong Huang Chun-zhi Tang Bao-ci Shan Shao-yang Cui jun-qi chen Shan-shan Qu Zheng Zhong Xin-sheng Lai Genevieve Zara Steiner School of Traditional Chinese Medicine Southern Medical University Guangzhou Guangdong Province China Traditional Chinese Medicine of the Second Clinical School Guangdong Medical College Dongguan Guangdong Province China Zhaoqing Medical College Zhaoqing Guangdong Province China Clinical Medical College of Acupuncture Moxibustion and Rehabilitation Guangzhou University of Chinese Medicine Guangzhou GuangdongProvince China Key Laboratory of Nuclear Analytical Techniques Institute of High Energy Physics Chinese Academy of Sciences Beijing China Shenzhen Hospital of Guangzhou University of Chinese Medicine Shenzhen Guangdong Province China Huarui Hospital of Southern Medical University Guangzhou Guangdong Province China National Institute of Complementary Medicine Western Sydney University Penrith NSW Australia

The acute effect of acupuncture on Alzheimer＇s disease,i.e.,on brain activation during treatment,has been ***,the effect of long-term acupuncture on brain activation in Alzheimer＇s disease is ***,in this study,we performed long-term needling at Zusanli（ST36）or a sham point（1.5 mm lateral to ST36）in a rat Alzheimer＇s disease model,for 30 minutes,once per day,for 30 *** rats underwent 18F-fluorodeoxyglucose positron emission tomography *** emission tomography images were processed with *** brain areas activated after needling at ST36 included the left hippocampus,the left orbital cortex,the left infralimbic cortex,the left olfactory cortex,the left cerebellum and the left *** the sham-point group,the activated regions were similar to those in the ST36 ***,the ST36 group showed greater activation in the cerebellum and pons than the sham-point *** findings suggest that long-term acupuncture treatment has targeted regulatory effects on multiple brain regions in rats with Alzheimer＇s disease.

关键词： nerve regeneration neurodegeneration acupuncture Zusanli (ST36) Alzheimer's disease long-term treatment positron emissiontomography rat mechanism targeting effect compensation multi-target regulation neural regeneration

Acupuncture for ischemic stroke:cerebellar activation may be a central mechanism following Deqi

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Neural Regeneration Research 2015年第12期10卷 1997-2003页

作者： Miao-keng Li Yu-jie Li Gui-feng Zhang jun-qi chen Ji-ping Zhang Ji qi Yong Huang Xin-sheng Lai Chun-zhi Tang School of Chinese Medicine Southern Medical University First Clinical Medical School Southern Medical University Zhaoqing Medical College Department of Rehabilitation Medicine the Third Affiliated Hospital of Southern Medical University College of Acupuncture and Massage Guangzhou University of Chinese Medicine

The needling sensation of Deqi during acupuncture is a key factor of influencing acupuncture *** studies have mainly focused on the brain function effects of Deqi in a physiological *** magnetic resonance imaging（f MRI）on the effects of acupuncture at Waiguan（SJ5）in pathological and physiological states is *** this study,12 patients with ischemic stroke received acupuncture at Waiguan（SJ5）and simultaneously underwent f MRI scanning of the brain,with imaging data of the activated areas *** on the patient＇s sensation,imaging data were allocated to either the Deqi group or non-Deqi *** the Deqi group,the activated/deactivated areas were the left superior temporal gyrus（BA39）/right anterior lobe of the cerebellum and left *** the non-Deqi group,the activated areas included the medial frontal gyrus of the right frontal lobe（BA11）,right limbic lobe（BA30,35）,and left frontal lobe（BA47）,while the only deactivated area was the right parietal lobe（BA40）.Compared with the non-Deqi group,the Deqi group exhibited marked activation of the right anterior lobe of the cerebellum and right limbic lobe（BA30）.These findings confirm that the clinical effect of Deqi during acupuncture is based on brain functional *** activation may be one of the central mechanisms of acupuncture in the treatment of ischemic stroke.

关键词： nerve regeneration traditional Chinese medicine acupuncture functional magneticresonance imaging ischemic stroke Brodmann area Waiguan (SJ5) acupoint Deqi non-Deqi 973Program neural regeneration