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
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A haem-sequestering plant peptide promotes iron uptake in symbiotic bacteria
Siva Sankari,Vignesh M. P. Babu,Ke Bian,Areej Alhhazmi,Mary C. Andorfer,Dante Avalos,Tyler A. Smith,Kwan Yoon,Catherine L. Drennan,Michael B. Yaffe,Sebastian Lourido,Graham C. Walker +11 more
TL;DR: In this article , the most extensively characterized NCR peptide, 24 amino acid NCR247, binds haem with nanomolar affinity and induces an ironstarvation response in rhizobia that results in iron import, which itself is required for nitrogenase activity.
Journal ArticleDOI
CRISPR-Cas9 and beyond: what’s next in plant genome engineering
Erin K. Zess,Matthew B. Begemann +1 more
TL;DR: This review will take care to discuss current plant genome engineering technologies, covering both well-established and up-and-coming tools, as well as describe potential and real-world applications.
Journal ArticleDOI
Enhanced soluble sugar content in tomato fruit using CRISPR/Cas9-mediated SlINVINH1 and SlVPE5 gene editing.
Baike Wang,Ning Li,Shaoyong Huang,Jiahui Hu,Qiang Wang,Yaping Tang,Tao Yang,Patiguli Asmutola,Juan Wang,Qinghui Yu +9 more
TL;DR: In this paper, two genes (SlinVinH1 and SlVPE5) were found to inhibit the accumulation of soluble sugar in tomato fruits and obtained two genes' knocked-out lines (CRISPR-invinh1 or CRISPRvpe5) using CRisPR/Cas9.
Journal ArticleDOI
CRISPR Tools for Physiology and Cell State Changes: Potential of Transcriptional Engineering and Epigenome Editing.
Christopher T. Breunig,Anna Köferle,Andrea M. Neuner,Maximilian F. Wiesbeck,Valentin Baumann,Stefan H. Stricker +5 more
TL;DR: The molecular basis of DNA targeting tools are discussed and the potential of these new technologies are reviewed before a summary of how these have already been used for the manipulation of cellular states and hypothesize about future applications are summarized.
Journal ArticleDOI
Advances in Delivery Mechanisms of CRISPR Gene-Editing Reagents in Plants
TL;DR: This review focuses on exploring delivery mechanisms categorized into Agrobacterium-mediated delivery and breakthroughs, particle bombardment-based delivery of biomolecules and recent improvements, and protoplasts, a versatile system for gene-editing and regeneration in plants.
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Multiplex Genome Engineering Using CRISPR/Cas Systems
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Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,F. A. Ran,David Benjamin Turitz Cox,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +10 more
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