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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CRISPR/Cas9‐mediated tetra‐allelic mutation of the ‘Green Revolution’ SEMIDWARF‐1 (SD‐1) gene confers lodging resistance in tef (Eragrostis tef)
Getu Beyene,Raj Deepika Chauhan,Justin Villmer,Nada Husic,Ning Wang,Endale Gebre,Dejene Girma,Solomon Chanyalew,Kebebew Assefa,Girma M. Tabor,Malia A. Gehan,Michael McGrone,Meizhu Yang,Brian Lenderts,Christine Schwartz,William J. Gordon-Kamm,Nigel J. Taylor,Donald J. MacKenzie +17 more
TL;DR: In this article , the authors used CRISPR/Cas9 to introduce knockout mutations in the tef orthologue of the rice SEMIDWARF•1 (SD•1) gene to confer semidwarfism and ultimately lodging resistance.
Journal ArticleDOI
An Extensible Vector Toolkit and Parts Library for Advanced Engineering of Plant Genomes
James Chamness,Jitesh Kumar,Anna J. Cruz,Elissa Rhuby,Mason J. Holum,Jon P. Cody,Redeat Tibebu,Maria Elena Gamo,Colby G. Starker,Feng Zhang,Daniel F. Voytas +10 more
TL;DR: A universal Golden Gate cloning toolkit for vector construction is described, compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits.
Journal ArticleDOI
Alfalfa (Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate.
Susan S. Miller,Melinda R. Dornbusch,Andrew Farmer,Raúl Huertas,Juan J. Gutierrez-Gonzalez,Nevin D. Young,Deborah A. Samac,Shaun J. Curtin +7 more
TL;DR: A set of novel alfalfa plants that hyper-accumulate Phosphate ion at levels three- to six-fold higher than wild-type is described, which will have practical applications reclaiming Pi from contaminated or enriched soil or be used in conservation buffer strips to protect waterways from Pi run-off.
Journal ArticleDOI
Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins.
Park Jongjin,Jiyoung Yoon,Daekee Kwon,Mi-Jung Han,Sunmee Choi,Slki Park,Junghyuk Lee,Kiwook Lee,Jaehwan Lee,Seung-Hee Lee,Kyung-Sun Kang,Sunghwa Choe,Sunghwa Choe +12 more
TL;DR: In this article, the authors created chimeric SpyCas9 proteins fused with the 5′to-3′ exonuclease Recombination J (RecJ) or with GFP and demonstrated that transfection of the pre-assembled ribonucleoprotein of the two chimeric proteins into human or plant cells resulted in greater targeted mutagenesis efficiency up to 600%.
Journal ArticleDOI
Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds
TL;DR: The results suggest that CNT-based delivery of plasmid DNA appears promising for in planta transformation, and further optimization can enable high-throughput gene editing to accelerate functional genomics and crop improvement activities.
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Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,Le Cong,F. Ann Ran,F. Ann Ran,David M. Cox,David M. Cox,Shuailiang Lin,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +15 more
TL;DR: The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage as discussed by the authors.
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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