Recipient Organization
TEXAS TECH UNIVERSITY
(N/A)
LUBBOCK,TX 79409
Performing Department
Plant and Soil Science
Non Technical Summary
Genomic information is rapidly being generated not only for major crops such as maize, wheat and rice, but also for other economically important crops including common bean, chili pepper, papaya, grapevine, etc. The increasing amount of information about plant genomes has allowed the use of different strategies, such as genome wide association studies, comparative genomics and use of mutant and mapping populations, to identify genes that contribute to different traits important for plant productivity. To validate candidate genes in most cases it is necessary to produce knockout or altered mutants of the gene of interest. The development of the CRISP/Cas9 technology that allows to edit genetic information to produce knockout, point mutations, changes in promoter strength (promoter bashing), DNA insertions, and gene replacements, has changed the way we do biology. Successful application of gene editing has been reported for many crop plants including maize, rice, soybean, cotton, among several others, but the tissue culture step to produce gene edited plants is time consuming and limited to the few laboratories that have the expertise in cell culture to regenerate these crop plants. To overcome the tissue culture hurdle to produce gene edited plants, we propose to design and develop a tissue-culture independent gene editing system that should be generally applicable to most broad leave crops, such as cotton, soybean, common beans, sunflowers, etc., and simple enough that could be carried out in a common greenhouse by people with basic training in horticulture or other plant science areas. The rational of this proposal is to use natural long-distance RNA mobility to supply cells in the shoot apical meristem with the RNAs required for gene editing without the need of the target cells to be directly subjected to the process of DNA, RNA, protein delivery, or the regeneration process. If we transfect leaf or cotyledon cells with the genes required to produce the editing machinery and only the RNAs move into meristematic cells, we will prevent undesirable DNA insertions, genetic and epigenetic alterations in the genome of the target cells that are often associated with the Agrobacterium-mediated transformation processes or abnormal developmental processes such as de novo meristem formation from differentiated cells. Gene-edited cells will still be part of the normal apical meristem, which later will rise to gametes that will produce gene edited seed under normal greenhouse conditions.
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
(N/A)
Developmental
50%
Goals / Objectives
General Objective: Design of a system to efficiently edit shoot apical meristem cells via a long-distance transport of the RNAs required for genome editing.Specific Objectives:1) Identification of an RNA motif capable of efficiently rendering long-distance mobility and entry into SAM cells encoding Cas9 and guide-RNAS. To achieve this objective, we will first examine the capacity of the UTRs of CENTRORADIALIS and GAI mRNAs, the 102 bases 5'UTR of FT mRNA movement motifs to efficiently mediate long-distance transport and entry into the SAM of large RNAs. We will also test the efficiency as zip-code elements of tRNAMet (TAIR No. At5g57885) and tRNAGly (TAIR No. At1g71700), which have been shown to be present in phloem sap and be capable of mobilizing mRNAS into meristems and flowers (Zhang et al., 2016). We will first produce a construct encoding a mCherry-GUS and mCherrry-Cas9 reporter gene fusions which will be linked to the different zip-code elements to test their capacity to mediate the long-distance transport and entry into the SAM of large mRNAs (Figure 3). The mRNAs will be produced using a binary vector containing a geminiviral origin of replication to amplify the DNA template for transcription and a gene construct to express the mCherry-GUS and mCherrry-Cas9 mRNAs under control of the double CaMV35S promoter and the terminator of the heat shock protein (HSP) gene, which has been shown to increase gene expression in several plants (Hirai et al., 2011).2) Design of constructs to produce the different components of the gene editing machinery with the signal to be transported into and process in SAM cells.To ensure that the Cas9 and the gRNAs are produced in the same cells of the SAM, we will use a polycistronic RNA containing the coding sequence of Cas9 and the sgRNA(s). The design requires the polycistronic RNA to be processed in a way that a Cas9 mRNA with a poly-A tail and mature sgRNAs are liberated. Multiple sgRNAs to target the same gene can be included in the polycistronic RNA to enhance gene editing. With that purpose, we will build the construct illustrated in figure 3. The construct contains the following components in 5' to 3' order: 1) the long intergenic region loop (LIR) of BeYDV to amplify the number of copies of the T-DNA as is commonly used in agroinfiltration experiments to enhance the levels of transcription; 2) a double CaMV35S promoter to achieve a very high level of transcription of the Cas9/sgRNA polycistronic cassette (Kay et al., 1987); 3) a zip-code sequence, i.e. the 102 nt 5' UTR of the Arabidopsis FT transcript; 4) the Cas9 coding sequence with codon optimization for dicots; 5) a synthetic poly-A tail, which will promote further polyadenylation and mRNA export from the nucleus upon processing of the polycistronic RNA by Cys4 (Dower et al., 2004); 6) a Cys4 recognition and cleavage sequence; 7) the sequence of the sgRNA(s) flaked by another Cys4 processing sequence; 8) and a double terminator of the Arabidopsis heat shock protein terminator and the Nos terminator and polyadenylation sequence, that has been shown to enhance transcript accumulation probably preventing transcriptional readthrough that reduces transcript stability (Yamamoto et al., 2018), and 9) the SIR, replicase gene and a second long intergenic region loop to complete the replication machinery of BeYDV. If a zip-code element other than the FT movement signal is found to be more efficient for long-distance transport and entry into de SAM, it will be placed in the polycistronic RNA after the last Cys4 cleavage sequence but before the transcription terminator sequences. 3) Delivery of Cys4 and Cas9/gsRNA into the SAM cells.For the processing of the Cas9/sgRNA polycistronic transcript, the presence in the same cells of Cys4 is required. However, it is probably not desirable to produce both the Cas9/sgRNA and the Cys4 transcripts in the same cells because processing would probably take place rapidly and before long-distance transport. Therefore, we will produce the Cas9/gsRNA and Cys4 transcripts in different leaves and at different timepoints. This will increase the probability that Cys4, upon translation in SAM cells, will process the Cas9/sgRNA transcript to produce Cas9 and mature sgRNAs for gene editing. To test when and where is most convenient to express Cys4 to be able to effectively provide Cys4 nuclease in SAM cell for the processing of the Cas9/sgRNA transcript, we will use a test system based on the requirement of a poly-A tail for efficient translation. We will test the following constructs: 1) a GFP or mCherry coding sequence followed by a ribozyme and its target site, and the Nos 3'end; the ribozyme will release a transcript lacking poly-A tail, 2) the fluorescent protein coding sequence followed by an artificial poly-A tail, a Cys4 processing signal and ribozyme and its target site that will release a transcript lacking a natural poly-A tail at the 3' end of the transcript, 3) a control having the fluorescent protein coding sequence followed by the Nos terminator and polyadenylation sequence. All constructs will include an RNA mobility signal or zip-code element such as the 102 nt FT movement motif.4) Selection of tissues to produce the RNAs required for gene editing.We will use agroinfiltration of leaves to produce the Cas9/sgRNA and Cys4 transcripts, which we expect to move via the phloem into SAM where meristematic cells would be subjected to gene editing. Agroinfiltration will be done on the abaxial side of leaves using a needleless syringe (different leaves or different areas of the same leave for each construct). For each Agrobacterium strain (Cas9/sgRNA or Cys4) we will test i) one, two and three agroinfiltrations, once every 24 h in the same leaf; ii) one, two or three simultaneous agroinfiltrations on the same leaf; and iii) two different leaves agroinfiltrated simultaneously one to three times or agroinfiltrated one 24 h after the other. N. benthamiana plants will be agroinfiltrated at the 4, 6 and 8 leaf stages. After agroinfiltration treatments, plants will be allowed to grow and the appearance of white photobleached sectors will be monitored. If white sectors are detected, derived from gene editing of the two PSD alleles in tobacco and 4 alleles in cotton, we will follow to seed production. At Texas Tech University, we have state-of-art greenhouse facility that in addition to light and temperature control, has a system to control light spectrum and light quality to modulate/accelerate the flowering time. Using this greenhouse system, we can have three to four cycles of N. benthamiana per year. Thus, we will have no problem to do experiments all year round and accelerate flowering when needed.5) Gene editing confirmation.We expect to observed white sectors in leaves that form after the DNA transfection processes either in greenhouse plants or germinating seeds, which size will depend on the number of cells in the SAM harboring edited PDS or CHL1 genes. However, if only one of the alleles has been edited, we will not see white sectors even do one of the two copies of the gene is mutated. Therefore, whether we see or not white sectors we will collect tissue of the apical tip of the plant from several plants, amplify the regions of the PDS or CHL1 genes and sequence the amplicon using an Illumina MySeq platform.
Project Methods
-MethodsAgrobacterium strains and agroinfiltrationFor agroinfiltration experiments we will test two A. tumefaciens strains, C58C1 and AGL1. Plasmids will be mobilized into A. tumefaciens strains via electroporation (Dower et al., 1988). Recombinant Agrobacterium will be prepared for infiltration using a modified protocol of Sainsbury and Lomonossoff (ref) [32]. Briefly, a single colony of recombinant bacteria will be inoculated into liquid LB media (10g/L tryptone, 5g/L yeast extract; 10g/L NaCl, pH 7) containing kanamycin (100mg/L) and rifampicin (50mg/L). Cultures will be grown overnight at 28°C with shaking. Bacteria will be concentrated by centrifugation (14,000g for 5min) and resuspended to an OD600 = 0.5 in MMA (10mM MES pH 5.6, 10mM MgCl2, 500μM acetosyringone) supplemented with 5 uM lipoid acid as recommended by Norkunas et al. (2018) to maximize transient expression. For transient expression, bacteria will be delivered by "syringe agroinfiltration" into the abaxial side of leaves of 2, 4 and 8-week old greenhouse grown plants-month using a blunt tipped plastic syringe and applying gentle pressure. The same agroinfiltration procedure will be applied for N. benthamiana and cotton, as good results have been reported for both species using similar protocols (Gu at al., 2014; Norkunas et al., 2018).Particle bombardmentEmbryos or cotyledons will be bombarded using a conventional PDS-1000/He System particle bombardment equipment from Biorad with an Hepta devise to optimize particle distribution. For transient expression in leaves of greenhouse plants a hand-held Helios Gene Gun System from Biorad. DNA coating and bombardment procedures will be essentially those described by Cabrera-Ponce, Vegas-Garcia and Herrera-Estrella (1995) with some modifications: 50 ml M-17 tungsten particles(15 mg/ml), 10 ml DNA (1ug/ml), 50 ml 2±5mCaCl and 20 ml of 1 mM spermidine are mixed in sequential order, then briefly sonicated. The mixture is centrifuged at10 000 rpm for 10 sec. The supernatant is discarded, and the pellet resuspended in 100 % ethanol. After a short sonication, aliquots are delivered onto the center of each macrocarrier. The tissue is bombarded twice at 1200 psi (4 h interval between bombardments). DNA coated particles for the Gen Gun system will be prepared using the same protocolVector constructionAll vectors will be constructed using the Golden Gate (GG) Strategy (Engler and Marillonnet 2014). The different components of each construct will be synthesized and inserted into L0 vectors from the GG Plant Toolkit (Engler et al., 2014). Then we will perform the L1 synthesis reaction and finally clone into an L2 GG binary vector containing the BeYDV origin of replication.