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Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases

Molecular Plant 2024年第5期17卷 824-837页

作者： Tom Schreiber Anja Prange Petra Schafer Thomas Iwen Ramona Grutzner Sylvestre Marillonnet Aurelie Lepage Marie Javelle Wyatt Paul alain tissier Department of Cell and Metabolic Biology Leibniz Institute of Plant BiochemistryWeinberg 306120 Halle(Saale)Germany Limagrain Centre de RechercheRoute d’EnnezatCS 9012663720 ChappesFrance

In plants and mammals,non-homologous end-joining is the dominant pathway to repair DNA doublestrand breaks,making it challenging to generate knock-in events.In this study,we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana.We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems.In Arabidopsis thaliana,fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants.In addition,we demonstrated stable and heritable knock-ins in wheat in 1%of the primary transformants.Taken together,our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.

关键词： homology-directed repair knockin gene replacement CRISPR-Cas 50 exonuclease plants

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nitiation of ER Body Formation and Indole Glucosinolate Metabolism by the Plastidia Retrograde Signaling Metabolite, MEcPP

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Molecular Plant 2017年第11期10卷 1400-1416页

作者： Jin-Zheng Wang Baohua Li Yanmei Xiao Yu Ni Haiyan Ke Panyu Yang Amancio de Souza Marta Bjornson Xiang He Zhouxin Shen Gerd Ulrich Balcke Steve P. Briggs alain tissier Daniel J. Kliebenstein Katayoon Dehesh Institute for Integrative Genome Biology and Department of Botany and Plant Sciences University of California Riverside CA 92521 USA Department of Plant Biology University of California Davis CA 95616 USA Department of Plant Sciences University of California Davis CA 95616 USA Division of Biological Sciences University of California San Diego La Jolla CA 92093 USA Department of Cell and Metabolic Biology Leibniz-lnstitute of Plant Biochemistry Halle Germany Present address: College of Agronomy and Biotechnology Southwest University Chongqing P.R. China Present address: Rice Research Institute Sichuan Agricultural University Changdu Sichuan 611130 China

Plants have evolved tightly regulated signaling networks to respond and adapt to environmental perturbations, but the nature of the signaling hub（s） involved have remained an enigma. We have previously established that methylerythritol cyclodiphosphate （MEcPP）, a precursor of plastidial isoprenoids and a stress- specific retrograde signaling metabolite, enables cellular readjustments for high-order adaptive functions. Here, we specifically show that MEcPP promotes two Brassicaceae-specific traits, namely endoplasmic reticulum （ER） body formation and induction of indole glucosinolate （IGs） metabolism selectively, via tran- scriptional regulation of key regulators NAIl for ER body formation and MYB51/122 for IGs biosynthesis）. The specificity of MEcPP is further confirmed by the lack of induction of wound-inducible ER body genes as well as IGs by other altered methylerythritol phosphate pathway enzymes. Genetic analyses revealed MEcPP-mediated COil-dependent induction of these traits. Moreover, MEcPP signaling integrates the biosynthesis and hydrolysis of IGs through induction of nitrile-specifier protein1 and reduction of the sup- pressor, ESM1, and production of simple nitriles as the bioactive end product. The findings position the plastidial metabolite, MEcPP, as the initiation hub, transducing signals to adjust the activity of hard- wired gene circuitry to expand phytochemical diversity and alter the associated subcellular structure required for functionality of the secondary metabolites, thereby tailoring plant stress responses.

关键词： glucosinolates ER body retrograde signaling MEP pathway MEcPP stress

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A focus on the European hamster to illustrate how to monitor endangered species

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Integrative Zoology 2019年第1期14卷 65-74页

作者： Florian KLETTY Mathilde tissier Charlotte KOURKGY Alexandre ZAHARIEV Nicolas CHATELAIN Julien COURTECUISSE Yvon LE MAHO Caroline HABOLD University of Strasbourg CNRSIPHCStrasbourgFrance National Hunting and Wildlife Agency ONCFSGerstheimFrance and 3Clinique VétérinaireColmarFrance

Agriculture intensification,marked by the generalization of crop monoculture,by the increase in plot size and by the reduction of plant diversity,has led to huge decline in wildlife in European farmlands.In such habitat,re-search has long been biased towards birds and invertebrates,while very few studies have investigated the ef-fect on small mammals.Considering the European hamster,Cricetus cricetus,we therefore review the different techniques that can be used to investigate the impact of environmental changes and conservation measures in small and endangered wild mammals.We suggest that only a multidisciplinary approach will allow exploration of these effects,combining experimental laboratory work on captive-bred animals with the monitoring of wild individuals.In particular,individual energy balance has to be investigated and measured as accurately as possi-ble,through either biochemical or bio-logging techniques.It is,indeed,the most affected physiological trait in a changing environment,as it determines both the reproductive output and the survival of the individual.We also discuss the inconvenience of capture-release approaches for such an endangered species and emphasize the dis-turbance that experimental protocols could impose on the hamster.

关键词： conservation measures data loggers degree of invasiveness energy balance metabolism

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1.5T MR的大兔模型中基质缺血与左心室重构的关系

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国际医学放射学杂志 2010年第4期 398-399页

作者： N.Mansencal R.tissier J.F.Deux B.Ghaleh N.Couvreur M.Rienzo 高飞 CMR Department Institut Hospitalier Jacques Cartier6 Avenue du Noyer Lambert91300 MassyFrance

目的心脏MR对比增强成像(CMR)对大型动物梗死面积的评估已被证明其可行性,但是研究和随访受到限制。本研究目的:①证实CMR在评估兔子再灌注心肌梗死(MI)模型的心肌风险

关键词：心室重构兔子再灌注 MR 舒张末期

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0CT测量肾脏长度用于预测活体肾供者的单侧肾GFR

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国际医学放射学杂志 2017年第2期 240-240页

作者： F.Gaillard P.Pavlov A.M.tissier B.Harache D.Eladari M.O.Timsit 石冰 AP-HP Hpital Necker-Enfants MaladesRenal Trans-plantation DepartmentParis Descartes University

摘要目的活体肾供者的筛选需要闪烁扫描测定单侧肾的肾小球滤过率(GFR)。为了明确CT对于评估单侧肾GFR的作用,将闪烁扫描与CT测定单侧GFR进行比较,评

关键词：单侧肾闪烁扫描放射性同位素扫描 GFR 活体肾脏尺寸长度供者 CT

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前列腺癌:实时剪切波弹性成像诊断性能

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国际医学放射学杂志 2015年第3期 285-285页

作者： J.M.Correas A.M.tissier A.Khairoune V.Vassiliu A.Méjean O.Hélénon 柯勤兵 Department of Adult Radiology Necker University Hos pital

目的前瞻性评估实时超声(US)剪切波弹性成像(SWE)在前列腺特异性抗原水平高和(或)升高、和(或)伴有直肠指检结果异常的病人中对于外周带前列腺癌的诊断性

关键词：诊断性能外周带病人 SWE 剪切波弹性成像前列腺癌

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