Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
School of Plant and Environmental Sciences
Non Technical Summary
JustificationProblem StatementTraditional crop breeding programs, using breeder selection of desirable traits found through introgression of wild relatives or derived from random mutagenesis through irradiation or treatment with mutagenic chemicals, is a time-consuming and labor-intensive process. Transgenic approaches, using the insertion of foreign DNA into the pant genome, can greatly shorten the time and effort required to introduce new traits into a crop lineage. However, transgenic crops are considered genetically modified organisms (GMO) and must undergo lengthy and expensive regulatory processes before they can be introduced onto the market. With the advent of new breeding technologies, such as CRISPR/Cas9 and related gene-editing methods, direct modification of crop genomes, as opposed to transgenesis, has become the way of the future for advanced breeding techniques in agriculture. These new tools have opened avenues of both fundamental and translational research that were previously inaccessible. The use of site-specific nucleases together with DNA repair templates has now made possible the precise manipulation of the genome to effect specific nucleic acid substitutions, insertions, or deletions. Thus, we can now alter the plant's own genome in order to modify crop traits to be more desirable for the growers, processors, and/or consumers. Furthermore, the ability to apply this technology through non-transgenic methods, i.e. without the insertion of the gene-editing tools into the plant genome, can avoid costly and time-consuming regulatory hurdles and accelerate the introduction of new crop lines to the ag market. The two biggest impediments to implementation of gene-editing technology in many agriculturally relevant crops are the low success rates and efficiencies of precise genetic modification and regeneration of true-to-type plants from tissue culture. Therefore, the project goal is to achieve more efficient ways to implement new breeding technology through the development of effectual gene-editing and plant regeneration methods. The general impact will be to speed up the application of new breeding technologies and to make them more broadly applicable to a variety of crop species.Relevance to advancing VA/Region and the U.S.Plant crops (soybeans, corn, wheat, tobacco, peanuts) contribute over $564M to Virginia's bio economy. Fruits and vegetables (e.g. apples, tomatoes, grapes, potatoes) contribute an additional $103M (2015 USDA NASS and ERS data). Virginia's agricultural industry is the largest private industry in the commonwealth, accounting for over $70B annually and employing more than 344,000 people. The development of more efficient methods in new breeding technologies will affect both the region of Virginia and the US as a whole. Economically, it can foster the startup of new ag biotech companies and associated businesses. Agriculturally, it can aid in the development of crops with altered traits (e.g. compatibility with modern weed-management practices, enhanced nutrition, or increased shelf-life) that benefit growers, processors, and consumers. Environmentally, it could help in the development of more sustainable crops and growing methods.ApproachRegeneration of plants from single cells is not trivial. Although there are many reports of successful regeneration from protoplasts, the methods employed and success rates differ substantially from lab-to-lab, species-to-species, and even between cultivars of the same species. The aim of this work includes finding ways to make regeneration more reliable and applicable in different germplasm. Having the ability to implement specific genome edits directly in elite germplasm, as opposed to lab strains selected to be amenable to tissue culture, would greatly speed up the breeding process and introduction of new crop lines. A better understanding of the regeneration process at a molecular level will allow us to manipulate the mechanisms involved to enhance and apply the process in otherwise recalcitrant systems. Regeneration is closely linked to embryogenesis and the overexpression of factors involved in embryo development and meristem maintenance is being used commercially to enhance regeneration from tissue culture. This approach is only beginning to be developed and has tremendous potential for broad applicability and direct impact on crop improvement strategies to cope with an increasing demand for food-, feed-, and bioenergy-crops in a world that requires continued adaptation to a changing climate.Gene-editing technologies for developing new crop varieties through genetic modification utilize the cellular protoplasm of the living crop plants. This is in contrast to the use of transgenic transformations, where foreign materials were introduced into the genetic material of familiar crops. Although there has been a continued use of protoplasts in fundamental research (e.g. for transient transformation assays to study protein localization, protein-protein interactions, enzymatic activity, and transcription factor-target interactions), the use of protoplasts for the purpose of genetic modification was mostly laid by the wayside upon the introduction of Agrobacterium tumefaciens mediated delivery of foreign DNA. There has been relatively little research dedicated to development of protoplast regeneration systems since the 1990's. The arrival of CRISPR/Cas9 technology has led to a resurgence of interest in protoplasts as a means to deliver gene-editing tools to plant cells. Protoplasts have the advantage that, with acquired expertise, large numbers of cells can be coaxed to express proteins-of-interest at relatively high efficiency (10-80%), compared to particle bombardment- or A. tumefaciens-mediated transformation (<1%) where most of the cells in the treated callus or explant are inaccessible to the particles or bacterial vector. Moreover, transient transformation, especially when delivering expressed tools in the form of mRNA or protein, precludes or greatly reduces the chances that foreign DNA is randomly integrated into the host genome, which would lead to an undesirable transgenic outcome. Importantly, the use of protoplasts also allows flexibility in the quantity and quality of DNA repair template delivered in conjunction with site-specific nucleases, these are crucial factors in the process of precise gene editing. Improvement of precise gene editing efficiency is essential, especially when targeting non-selectable traits, i.e. gene edits for which screening by DNA sequencing will be required to identify successfully edited events. A success rate of one in a thousand is the minimum needed to be able to perform manual screening, but increased efficiency will greatly reduce the labor requirements and enable greater throughput.
Animal Health Component
(N/A)
Research Effort Categories
Basic
25%
Applied
(N/A)
Developmental
75%
Goals / Objectives
Major Goals and ObjectivesThis project has two main goals:Goal 1. Develop an experimental system to characterize embryogenesis-related TFs that can enhance regeneration from protoplasts.1.1 Clone a suite of embryogenesis-related TFs from Arabidopsis thalianaThe first objective is to come up with suite of twenty to thirty TFs known to be involved in embryogenesis; this is a number well-suited to the TARGET system, as recently demonstrated by Brooks and coworkers (Brooks et al., 2019). Among the selected TFs will be members of the AP2 family of TFs, such as BABYBOOM and EMBRYOMAKER, that have previously been implicated in embryogenesis and shown to induce ectopic embryo formation when over-expressed (Tsuwamoto et al., 2010, El Ouakfaoui et al., 2010). TFs closely related to known embryogenic regulators or known to be downstream of known embryogenic regulators will also be included.1.2 Optimize the TARGET system for the identification of direct targets of embryogenic TFsThe development of a system to characterize the gene regulatory networks governed by embryogenic TFs will allow us to strengthen our fundamental understanding of the process. The hierarchy and interplay between the different TFs that have already been shown to be involved in embryogenesis will point us to which of our candidates may be most appropriate to start tests for enhancement regenerative capacity of protoplasts. Furthermore, such analyses will likely lead us to new candidates for further study.1.3 Establish protoplast regeneration systems to test the ability of embryogenic TFs to enhance regeneration efficiencyThere are published methods available for the regeneration of whole plants from protoplasts in Arabidopsis, e.g. by Dovzhenko and coworkers (Dovzhenko et al., 2003). We will work to establish these procedures in the lab using the Arabidopsis thaliana Col-0 accession. It is important to be able to repeat these methods in a reliable and routine manner, so that we can ascertain whether the transient expression of our candidate embryogenic TFs can reproducibly enhance the regeneration efficiency.Goal 2. Set up standard operating procedures to implement gene-editing technology through transient transformation and regeneration of protoplasts.2.1 Establish high-efficiency InDel formation by CRISPR/Cas9 in protoplastsHigh gene-editing efficiency is crucial to the development of platform for the genetic modification of crop traits, especially when targeting non-selectable traits. To achieve this goal, it is of great value to have a model system to test various strategies before employing them in applications with defined targets in specific crops. Firstly, we will set up a method to readily test transformation efficiency. Secondly, we will develop a method to rapidly test InDel formation by CRISPR/Cas9 in protoplasts.2.2 Establish precision gene editing in protoplastsPrecision gene editing, i.e. the targeted substitution of specific nucleotides within a gene sequence, can be achieved in several ways. Firstly, it can be done by co-delivery of a site-specific double-stand breaker (e.g. TALEN or Cas9) and a DNA-repair template (Sauer et al., 2016). Secondly, we can make use of a base-editing Cas9 variant employing cytidine deaminase fusions (Kim et al., 2017). Lastly, the recently developed prime-editing technology uses a fusion of Cas9 with a reverse-transcriptase that synthesizes the DNA-repair template in situ (Anzalone et al., 2019). These technologies allow for the alteration of gene function, as opposed to a knock-out of gene function achieved by InDel formation. Each of these methods have different advantages are more or less suited for specific applications. All will be introduced to the lab and optimized to efficiencies suitable for the successful regeneration of gene-edited plants without the use of selection. Various cloned constructs are available to the academic community, e.g. through addgene.com.2.3 Regeneration of plants from gene-edited protoplasts The enhanced and more universal protoplast regeneration strategies developed under Goal 1 will be combined with the gene-editing protocols established under objectives 2.1 and 2.2 to achieve reproducible, high-efficiency, and non-transgenic production of gene-edited plants. To this end, standard operating procedures, proof-of-concept systems, molecular and phenotypic validation assays, plant propagation, and germplasm storage methods will need to be in place.
Project Methods
Experimental Procedures1.1Cloning TFsIn order to clone embryogenic TFs, we need to extract RNA from tissues where they are expressed. To this end, we will initiate the culture of embryogenic callus from Arabidopsis. Primers will be designed based on the sequences as deposited on arabidopsis.org and made compatible with the Gateway cloning system (Karimi et al., 2007) to get them into pDONR plasmids; the TARGET system is Gateway-compatible (Figure 5). The lab also has access to a number of other Gateway-compatible plant expression vectors that can subsequently be used to study the function of our selected TFs.1.2Optimizing TARGETOne important aspect of employing the TARGET system to study embryogenic TFs will be the choice of tissue from which the protoplasts used for transformation will be derived. So far, the TARGET system has only been deployed in protoplasts obtained from the roots of Arabidopsis seedlings (Bargmann et al., 2013, Para et al., 2014, Brooks et al., 2019); a protoplast source where these factors are normally expressed would be better suited. To this end, we will develop the use of protoplasts derived from embryogenic callus with the TARGET system.The dexamethasone and cycloheximide treatments of protoplasts transformed with the TARGET system will require further optimization. Time course and concentration curve tests will be performed to find the optimal treatment parameters for the identification of directly regulated genes.Lastly, we will need to develop an analysis pipeline for the high throughput transcriptomic data. RNAseq libraries will be produced in-house and the sequencing will be outsourced. Analysis of the data will be performed in-house with the help of expert colleagues (Dr. David Haak and Dr. Song Li, VT SPES). Methods will be developed to construct gene-regulatory networks in silico in order to analyze the hierarchy and complementarity of embryogenic master regulators.1.3Establishing a regeneration systemMethods will be developed to transiently express TFs using the pBeaconRFP vector (Bargmann & Birnbaum, 2009). We will also test whether protoplasts can be regenerated after sorting them by FACS. Additionally, we will test the feasibility of transforming the protoplasts with multiple constructs at once. Lastly, methods for DNA-free expression in protoplasts will be developed, i.e. transformation of protoplasts with mRNA or purified protein instead of plasmid DNA.Furthermore, regeneration of protoplasts derived from various tissues will be established. Such tissues include true leaves, roots, and (embryogenic) callus. Subsequently, we will set up protoplast regeneration systems in relevant crop species, focusing initially on horticultural specialty crops, e.g. potato and heirloom tomato varieties. The aim will be to enable assessment of candidates for the potential to enhance protoplast regeneration.2.1Establishing high-efficiency InDel formationAs a readily quantifiable measure for transformation efficiency, protoplasts will be transfected with a GFP expression plasmid. PEG-mediated delivery will be the method of preference, and electroporation will be the back-up strategy. Efficiency will be assayed cytometrically, as validated by initial manual hemacytometer counts.To obtain a system that enables cytometric detection of InDel formation that leads to knockout of gene function, we will generate transgenic plants that (inducibly) express both green and red fluorescent proteins (GFP and mCherry). The efficiency of CRISPR/Cas9 constructs that target one of the two fluorescent protein sequences can then be assayed by measuring the ratio of red-to-green fluorescence in treated protoplasts cytometrically. This system will allow us to test various CRISPR/Cas9 constructs and delivery methods in a practicable and inexpensive manner. The system will be validated by molecular analysis of InDel formation efficiency. A T7 endonuclease 1 mismatch detection assay as well as alternate molecular assays (e.g. Tracking of Indels by Decomposition and Indel Detection by Amplicon Analysis) will be used when appropriate to get an accurate readout (Sentmanat et al., 2018). Initial experiments will be conducted with plasmid delivery of the Cas9 and guideRNAs (e.g. pRGE3210) and the switch to ribonucleoprotein or transcript delivery will be made once acceptable success rates with plasmid delivery have been achieved.2.2Establishing precision gene editingNext generation sequencing will be used to assay precise gene-editing success rates. In addition, the more rapid and high-throughput method using cytometric evaluation of BFP-to-GFP conversion will be established to evaluate different gene-editing tool delivery strategies. This will require the generation of transgenic BFP-expressing lines and the setup and validation of cytometric analysis protocols. The timing of the delivery and the amount of the gene-editing reagents used for the transformation will be tested systematically to determine the optimal procedure for high-efficiency gene editing using this rapid evaluation tool. The system will initially be developed in Arabidopsis and transitioned to relevant crop species as the work progresses.2.3Regenerating edited plants The regeneration of protoplasts will need to be tested on protoplasts treated with gene-editing tools. This may require the adaptation of regeneration and/or gene-editing techniques. As a proof-of-concept for gene knockout through InDel formation, aside from the GFP/mCherry described in objective 2.1, we will target the phytoene desaturase gene (PDS). Successful knockout of this gene function will lead to the development of regenerants without chlorophyll, which is more practicable to screen for in high numbers than the loss of fluorescence. As a proof-of-concept for precise gene edits, aside from the BFP-to-GFP conversion described in objective 2.2, we will target the introduction of herbicide tolerance through modification of the AHAS enzyme (Figure 4b). Successful precision edits of this gene will lead to resistance to AHAS-inhibiting herbicides, which is more practicable to screen for in high numbers than the gain of GFP fluorescence. Both of these concepts also do not require the generation of transgenic plants expressing fluorescent proteins, which may not be possible for all recipient species. Analysis of the genomic sequence in the target genes, either through qPCR, Sanger sequencing, or next-generation sequencing will be streamlined in order to obtain the capacity for high-throughput screening. Phenotypic screening of regenerated plants, both for off-types due to somaclonal variation and to assess the effect of specific genetic manipulations will require routine and reproducible culture and growth procedures. Selected successfully edited plants will need to be propagated and their germplasm consigned to long-term storage. This may require periodic regrowth and harvest of fresh seed (or propagules). All such procedures will be standardized, documented, and passed on to students, research assistants, postdocs, and other academic colleagues.All the work described above will be performed in the Bargmann lab in Latham Hall, where all the required equipment and facilities, including a FACS and growth chambers, are available at this time.