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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Journal ArticleDOI
Fast-TrACC: A Rapid Method for Delivering and Testing Gene Editing Reagents in Somatic Plant Cells.
TL;DR: Fast-TrACC (Fast Treated Agrobacterium Co-Culture) as discussed by the authors uses a luciferase reporter to monitor and calibrate the efficiency of reagent delivery to test molecular reagents transiently.
Journal ArticleDOI
Apoptotic-like PCD inducing HRC gene when silenced enhances multiple disease resistance in plants
Ajjamada C. Kushalappa,N. G. Hegde,Raghavendra Gunnaiah,Atul Sathe,Kalenahalli N. Yogendra,L. Ajjamada +5 more
TL;DR: In this article , a non-HR type or Apoptotic-Like PCD (AL-PCD) was reported in pathogen infected wheat and potato based on apoptotic-like DNA fragmentation.
Journal ArticleDOI
General guidelines for CRISPR/Cas-based genome editing in plants
Emre Aksoy,Kubilay Yıldırım,Musa Kavas,Ceyhun Kayıhan,Bayram Ali Yerlikaya,Irmak Çalik,İlkay Sevgen,Ufuk Demirel +7 more
TL;DR: This review summarizes the type of promoters, Cas proteins, recognition sequences, and terminators available for the development of knock-out and overexpression plant lines and provides a general guideline for theDevelopment of genome-edited plants from the design of sgRNAs to the selection of non-transgenic genome- edited T2 generation.
Book ChapterDOI
Genome editing in the context of seed research: How these novel biotechnology tools can change the future face of agricultural crop development
Chiara Forti,Carla Gualtieri,Andrea Pagano,Susana de Sousa Araújo,Alma Balestrazzi,Anca Macovei +5 more
TL;DR: This chapter intends to take into account the application of genome editing techniques focusing on seed quality traits, including aspects related to seed vigor, dormancy, germination, and features covering seed nutritional/biochemical composition to address current and future crop improvements.
Journal ArticleDOI
Advances in Barley Breeding for Improving Nitrogen Use Efficiency
Zhiwei Chen,Luli Li,Nigel G. Halford,Hongwei Xu,Linli Huang,Runhong Gao,Ruiju Lu,Chenghong Liu +7 more
TL;DR: In this paper , a review of recent progress in understanding barley's response to nitrogen nutrition, evaluation of NUE or low-nitrogen tolerance, quantitative trait loci (QTL) mapping and gene cloning associated with improving NUE, and breeding of nitrogen-efficient barley is summarized.
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Multiplex Genome Engineering Using CRISPR/Cas Systems
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