A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants
Tomas Cermak,Shaun J. Curtin,Javier Gil-Humanes,Radim Cegan,Thomas J. Y. Kono,Eva Konečná,Joseph J. Belanto,Colby G. Starker,Jade W. Mathre,Rebecca L. Greenstein,Daniel F. Voytas +10 more
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TLDR
An integrated reagent toolkit and streamlined protocols work across diverse plant species to enable sophisticated genome edits and it is demonstrated that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons.Abstract:
We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination Regulation of transcription is also possible A Web-based tool streamlines vector selection and construction One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs) For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters Mutagenesis can be further enhanced 25-fold by incorporating the Trex2 exonuclease Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare)read more
Citations
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Journal ArticleDOI
Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing.
Daniel Rodríguez-Leal,Zachary H. Lemmon,Jarrett Man,Madelaine E. Bartlett,Zachary B. Lippman +4 more
TL;DR: It is demonstrated that CRISPR/Cas9 genome editing of promoters generates diverse cis-regulatory alleles that provide beneficial quantitative variation for breeding that provide a foundation for dissecting complex relationships between gene-reg regulatory changes and control of quantitative traits.
Journal ArticleDOI
De novo domestication of wild tomato using genome editing
Agustin Zsögön,Tomas Cermak,Emmanuel Rezende Naves,Marcela Morato Notini,Kai H. Edel,Stefan Weinl,Luciano Freschi,Daniel F. Voytas,Jörg Kudla,Lázaro Eustáquio Pereira Peres +9 more
TL;DR: It is reported that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium, paving the way for molecular breeding programs to exploit the genetic diversity present in wild plants.
Journal ArticleDOI
Prime genome editing in rice and wheat
Qiupeng Lin,Yuan Zong,Chenxiao Xue,Shengxing Wang,Shuai Jin,Zixu Zhu,Yanpeng Wang,Andrew V. Anzalone,Andrew V. Anzalone,Andrew V. Anzalone,Aditya Raguram,Aditya Raguram,Aditya Raguram,Jordan L. Doman,Jordan L. Doman,Jordan L. Doman,David R. Liu,David R. Liu,David R. Liu,Caixia Gao +19 more
TL;DR: The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts through codon, promoter, and editing-condition optimization.
Journal ArticleDOI
Domestication of wild tomato is accelerated by genome editing.
Tingdong Li,Xinping Yang,Yuan Yu,Xiaomin Si,Xiawan Zhai,Huawei Zhang,Wenxia Dong,Caixia Gao,Cao Xu +8 more
TL;DR: This work introduced desirable traits into four stress-tolerant wild-tomato accessions by using multiplex CRISPR–Cas9 editing of coding sequences, cis-regulatory regions or upstream open reading frames of genes associated with morphology, flower and fruit production, and ascorbic acid synthesis.
Journal ArticleDOI
Applications of CRISPR–Cas in agriculture and plant biotechnology
Haocheng Zhu,Chao Li,Caixia Gao +2 more
TL;DR: The most important applications of CRISPR-Cas in increasing plant yield, quality, disease resistance and herbicide resistance, breeding and accelerated domestication, and prospective applications of this game-changing technology are discussed.
References
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Book ChapterDOI
Genetic Transformation of Wheat: Advances in the Transformation Method and Applications for Obtaining Lines with Improved Bread-Making Quality and Low Toxicity in Relation to Celiac Disease
Javier Gil-Humanes,Carmen V. Ozuna,Santiago Marín,Elena León,Francisco Barro,Fernando Pistón +5 more
TL;DR: Among the applications of genetic transformation, gene over-expression and posttranscriptional gene silencing (PTGS) are two strategies successfully used to enhance the wheat quality, and the baking quality of wheat, largely determined by the high molecular weight glutenin subunits (HMW-GS), is one of the most important targets for genetic transformation.