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      Molecular tools enabling pennycress ( Thlaspi arvense) as a model plant and oilseed cash cover crop

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          Summary

          Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run‐off on otherwise barren farmland. We demonstrate that pennycress can serve as a user‐friendly model system akin to Arabidopsis that is well‐suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring‐type Spring 32‐10 inbred line (1C DNA content of 539 Mb; 2 n = 14), identifying variation that may explain phenotypic differences with winter‐type pennycress, as well as predominantly a one‐to‐one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium‐mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR‐Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 ( FAE1 ) gene, thereby abolishing erucic acid production and creating an edible seed oil comparable to that of canola. We also stably transformed pennycress with the Euonymus alatus diacylglycerol acetyltransferase ( Ea DAcT ) gene, producing low‐viscosity acetyl‐triacylglycerol‐containing seed oil suitable as a diesel‐engine drop‐in fuel. Adoption of pennycress as a model system will accelerate oilseed‐crop translational research and facilitate pennycress’ rapid domestication to meet the growing sustainable food and fuel demands.

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          Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.

          Engineered nucleases can be used to induce site-specific double-strand breaks (DSBs) in plant genomes. Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error-prone non-homologous end-joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ. Cas9 can also be engineered to work as a nickase inducing single-strand breaks (SSBs). Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ-mediated mutagenesis in plants. We demonstrate the stable inheritance of nuclease-induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 2.5 up to 70.0%. Deep sequencing analysis revealed NHEJ-mediated DSB repair in about a third of all reads in T1 plants. In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740-fold. Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I-SceI. Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB-inducing enzymes. Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants. It should not only be applicable for HR-mediated gene targeting systems but also by the combined action of two nickases as DSB-inducing agents excluding off-target effects in homologous genomic regions. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.
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            Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

            Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.
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              Plant triacylglycerols as feedstocks for the production of biofuels.

              Triacylglycerols produced by plants are one of the most energy-rich and abundant forms of reduced carbon available from nature. Given their chemical similarities, plant oils represent a logical substitute for conventional diesel, a non-renewable energy source. However, as plant oils are too viscous for use in modern diesel engines, they are converted to fatty acid esters. The resulting fuel is commonly referred to as biodiesel, and offers many advantages over conventional diesel. Chief among these is that biodiesel is derived from renewable sources. In addition, the production and subsequent consumption of biodiesel results in less greenhouse gas emission compared to conventional diesel. However, the widespread adoption of biodiesel faces a number of challenges. The biggest of these is a limited supply of biodiesel feedstocks. Thus, plant oil production needs to be greatly increased for biodiesel to replace a major proportion of the current and future fuel needs of the world. An increased understanding of how plants synthesize fatty acids and triacylglycerols will ultimately allow the development of novel energy crops. For example, knowledge of the regulation of oil synthesis has suggested ways to produce triacylglycerols in abundant non-seed tissues. Additionally, biodiesel has poor cold-temperature performance and low oxidative stability. Improving the fuel characteristics of biodiesel can be achieved by altering the fatty acid composition. In this regard, the generation of transgenic soybean lines with high oleic acid content represents one way in which plant biotechnology has already contributed to the improvement of biodiesel.
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                Author and article information

                Contributors
                jcsedbr@ilstu.edu
                Journal
                Plant Biotechnol J
                Plant Biotechnol. J
                10.1111/(ISSN)1467-7652
                PBI
                Plant Biotechnology Journal
                John Wiley and Sons Inc. (Hoboken )
                1467-7644
                1467-7652
                25 October 2018
                April 2019
                : 17
                : 4 ( doiID: 10.1111/pbi.2019.17.issue-4 )
                : 776-788
                Affiliations
                [ 1 ] School of Biological Sciences Illinois State University Normal IL USA
                [ 2 ] School of Agriculture Western Illinois University Macomb IL USA
                [ 3 ] Department of Plant Biology University of Minnesota Saint Paul MN USA
                [ 4 ] Department of Biochemistry and Molecular Biophysics Kansas State University Manhattan KS USA
                [ 5 ] Center for Plant Science Innovation and Department of Biochemistry University of Nebraska‐Lincoln Lincoln NE USA
                Author notes
                [*] [* ]Correspondence (Tel 309‐438‐3374; fax 309‐438‐3722; email jcsedbr@ 123456ilstu.edu )
                Author information
                http://orcid.org/0000-0002-7277-1176
                http://orcid.org/0000-0002-4930-1627
                Article
                PBI13014
                10.1111/pbi.13014
                6419581
                30230695
                81fecdb9-49a4-4216-bdee-ded5c3fd218e
                © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 22 May 2018
                : 25 August 2018
                : 04 September 2018
                Page count
                Figures: 5, Tables: 1, Pages: 13, Words: 11207
                Funding
                Funded by: Walton Family Foundation
                Funded by: U.S. Department of Energy, Office of Science, OBER
                Award ID: DOE BER SC0012459
                Funded by: National Institute of Food and Agriculture, U.S. Department of Agriculture,
                Award ID: 2014‐67009‐22305
                Award ID: 2015‐67013‐22815
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                pbi13014
                April 2019
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.6.1 mode:remove_FC converted:15.03.2019

                Biotechnology
                pennycress,genome,domestication,triacylglycerol,crispr,transformation
                Biotechnology
                pennycress, genome, domestication, triacylglycerol, crispr, transformation

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