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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OtherDOI
The use of CRISPR/CAS9 as a reverse genetics tool to validate genome-wide association candidates
Journal ArticleDOI
Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-generated diallelic mutants reveal Arabidopsis actin-related protein 2 function in the trafficking of syntaxin PEN1
Peng Gao,Lian-Ju Qin,Hanh Nguyen,Huajin Sheng,Teagen D. Quilichini,Daoquan Xiang,Leon V. Kochian,Yangdou Wei,Raju Datla +8 more
TL;DR: This CRISPR/Cas9 multiplexing-mediated genome editing approach offers highly efficient simultaneous disruption of the two ARP2 alleles in GFP-PEN1-expressing lines, and a rapid method for performing live-cell imaging to facilitate the investigation of important plant–pathogen interactions using a well-established and widely applied GFP marker system.
Patent
CRISPR-CAS systems for genome editing
TL;DR: In this article, the Cas endonuclease was used for genome modification of a target sequence in the genome of a cell, using a novel Cas end-uclease, and the methods and compositions employed a guide polynucleotide/endonuclease system to provide an effective system for modifying or altering target sequences within the genome.
Journal ArticleDOI
The tomato calcium-permeable channel 4.1 (SlOSCA4.1) is a susceptibility factor for pepino mosaic virus.
Fabiola Ruiz-Ramón,Pascual Rodriguez-Sepulveda,P. Breto,Livia Donaire,Y Hernando,Miguel A. Aranda +5 more
TL;DR: In this article , a CRISPR/Cas9 slosca4.1 mutant was found to be resistant to pepino mosaic virus (PepMV) but not to tobacco mosaic virus or potato virus X.
Journal ArticleDOI
Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two
Jana Ordon,Niklas Kiel,Dieter Becker,Carola Kretschmer,Paul Schulze-Lefert,Johannes Stuttmann +5 more
TL;DR: In this article , the combination of guide RNA pairs and co-expression of the exonuclease TREX2 was used to increase editing efficiency in Arabidopsis without obvious negative effects.
References
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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
TL;DR: Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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