Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
Performing Department
Plant Science & Landscape Architecture
Non Technical Summary
CRISPR-Cas-based genome editing tools have been developed and widely used for crop improvement. However, CRISPR-Cas systems cannot be applied to edit organelle (chloroplast/plastid and mitochondrion) genomes since the guide RNAs cannot be effectively delivered into organelles. Recent studies showed that transcription activator-like effector (TALE)-based deaminases can induce precise base substitution in organelles. However, its low editing efficiency has hindered its application in plants. Our lab has recently developed CRISPR-Combo systems, which help enrich genome editing events by simultaneous activation of OsBBM1 in rice in a hormone-free tissue culture condition. Earlier, we also developed an effective TALE activation system. In this project, we will combine our expertise in these areas to develop efficient TALE-Combo systems to enrich plastid cytosine and adenine base editing events in rice. As a proof-of-concept, we will target the plastid-encoded genes, PsaA and PsbA, whose alteration directly affects the photosynthesis efficiency and crop yield. Plastid genome-edited plants, after segregation of the transgene in the nuclear genome, may be regulated as non-transgenic. Hence, our TALE-Combo organellar genome editing systems will not only accelerate crop engineering but also contribute to improving global food and nutrition security.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Goals / Objectives
Objective 1: Develop a highly potent TALE-Act2.0 system for nuclear gene activation.Five new TALE activation systems based on different activators (TALE-VP64, TALE-TAD, TALE-2×TAD, TALE-VPR, and TALE-TV) will be developed and assessed in rice protoplasts. By testing three different target genes including OsBBM1 with different TALE binding sites, we will benchmark their activation potency in rice protoplasts using qRT-PCR. We expect to develop an efficient TALE-Act2.0 system in plants, better than the TALE-Act system that we developed previously. The strong OsBBM1 activation conferred by TALE-Act2.0 should allow for hormone-free plant regeneration in rice tissue culture. This strategy will be used to demonstrate the CBE-TALE-Combo and ABE-TALE-Combo systems in Objective 2 and Objective 3 to enrich high efficiency plastid baes editing events.Objective 2: Develop an efficient CBE-TALE-Combo system to enrich the homoplasmic C-to-T editing in rice.To develop CBE-TALE-Combo system, the previously demonstrated TALE-DdCBE pair (Left-G1397N+Right-G1397C) enables C-to-T base editing will be employed and assembled with TALE activation components from Objective 1 into the same T-DNA vector. By targeting two rice chloroplast genes, PsbA and PsaA, respectively, and simultaneous activation of OsBBM1, we will generate two CBE-TALE-Combo T-DNA vectors. The two DdCBE pairs for genome editing only will be used as controls to edit the same genes (PsbA and PsaA). These four resulting T-DNA vectors will be first tested in rice protoplasts with a PEG method for transient assessment. Then Agrobacterium-mediated transformation will be used to generate stable rice lines for cpDdCBE and CBE-Combo T-DNA using medium with and without hormones, respectively.For each T-DNA vector, at least 30 T0 transgenic rice plants will be obtained to analyze targeted base editing efficiency. We expect CBE-TALE-Combo will generate significantly higher C-to-T base editing and enrich homoplasmic mutations in plastid genome than controls by activation of OsBBM1 with hormone-free medium.Objective 3: Develop an efficient ABE-TALE-Combo system to enrich the homoplasmic A-to-G editing in rice.To develop ABE-TALE-Combo system, the previous demonstrated TALED pairs (Left-G1397N+Right-G1397C-AD) enable A-to-G base editing will be employed and assembled with TALE activation components from Objective 1 into the same T-DNA vector. The target genes selected in the Objective 2 will be further used to evaluate the efficiency of A-to-G base editing. Similar to Objective 2, by targeting these two rice chloroplast genes, PsbA and PsaA, respectively, and simultaneous activation of OsBBM1, we will generate two ABE-TALE-Combo T-DNA vectors. The two TALED pairs will be used as controls by just targeting the same chloroplast genes without OsBBM1 activation. Protoplast-mediated transient assay and Agrobacterium-mediated stable transformation using both hormone-containing and -free medium will be carried out to compare the A-to-G efficiency between ABE-TALE-Combo and TALED vectors. We expect ABE-TALE-Combo will generate significantly higher A-to-G base editing and enrich homoplasmic mutations in rice plastid genomes than controls by activation of OsBBM1 on hormone-free medium.
Project Methods
Objective 1: Develop a highly potent TALE-Act2.0 system for nuclear gene activation.The gBlock gene fragments of different transcription activation domains (VP64, TAD (TAL Activation Domain), 2xTAD, VPR (VP64-p65-Rta), and TV (6xTAL-VP128)) will be synthesized and cloned into pYPQ127B, respectively. TALE-binding target sequence (14-18 bp) targeting the 150-350 bp upstream of transcription start site (TSS) of OsGW7 and OsBBM1 will be designed using TALE Effector Targeter (https://tale-nt.cac.cornell.edu/node/add/single-tale). The TALE assembly will be conducted based on the previously published protocol [18]. The assembled full TALE repeats will then be excised from the pZHY500 and cloned into pYPQ127B containing an activation domain between XbaI and BamHI sites, resulting in TALE activator vector. pYPQ127B plasmid contains a 2x35S promoter and a NOS terminator, which can directly control the expression of TALE repeats and activator fusion. For each TALE activator, we will design three-to five TALE repeats/target sites for each target gene, generating a total of 30-45 expression vectors for testing in rice cells.We will perform rapid analysis of these expression vectors using the rice protoplast assay established in our lab. Briefly, the TALE activator vectors expressing TALE repeats and activator fusion will be midi-prep with the Qiagen-Midi kit. Protoplasts will be prepared using 10-day-old etiolated seedlings of japonica rice variety Kitaake. Based on our experience, we expect > 85% transfection efficiency using the GFP-expression vector/marker. After 48 h incubation, the transfected protoplasts will be collected. Total RNA extraction, first-strand synthesis, and qRT-PCR will be carried out according to commercial kit instructions. The relative expression level will be calculated according to the 2-ΔΔCt method [19]. The expression data will be normalized against the expression level of the reference gene OsTublin. Three biological replicates will be performed for each vector. By testing five different transcription activators, we expect to benchmark and identify the most potent TALE activator, which will be named TALE-Act2.0, in reference to our earlier mTALE-Act system [17]. In addition, we expect to identify an assembled TALE-Act2.0 with activation of OsBBM1 gene of > 50-fold for hormone-free plant regeneration in Objectives 2 and Objective 3. If needed, we will screen more target sites by engineering the corresponding TALE repeats into the TALE-Act2.0 vector to meet this expected transcriptional activation potency.Objective 2: Develop an efficient CBE-TALE-Combo system to enrich the homoplasmic C-to-T editing in rice.To construct TALE-DdCBE pair (Left-G1397N+Right-G1397C), the gBlock gene fragments containing chloroplast target peptide (CTP) from maize ribulose bisphosphate carboxylase small subunit 2 with a triple FLAG-tag (Left TALE) or a triple HA-tag (Right TALE), rice-codon optimized split-DddAtox, and UGI will be synthesized by Integrated DNA Technology (IDT) with both KpnI and AatII restriction enzyme sites. These gBlocks will be digested and cloned into pYPQ121 between KpnI and AatII sites, separately, which can generate pYPQ121-1 (that contains cpDdCBE). The Left and Right TALE repeats targeting plastid genes PsaA and PsbA will be first assembled into pZHY500. Then, they will be excised from pZHY500 and ligated into pYPQ121-1 between XbaI and BamHI sites or between NheI and BglII sites based on our established protocol [14]. The final T-DNA vectors will be generated through Multisite Gateway reaction with pYPQ121-1-left&right (that contains left and right TALE repeats), pYPQ127B (that contains best TALE-Act2.0 activator for OsBBM1 activation) and a destination vector pYPQ203 (that contains the promoter to express the left and right TALE repeats for base editing). The resulting two CBE-TALE-Combo T-DNA vectors and the two control CBE-TALE vectors (without OsBBM1 activation) will be used to transform rice protoplasts to test the plastid editing efficiency (quantified by deep sequencing of PCR amplicons) and, if applicable, OsBBM1 activation (quantified by qRT-PCR).Once the editing activity of these vectors is confirmed in rice protoplasts, they will be transformed into rice calli using the Agrobacterium-mediated transformation which is also routinely done in our lab. Briefly, rice calli will be inoculated with Agrobacterium EHA105 trains harboring the corresponding binary vectors for three days, which will be subsequently washed and moved into selection medium with 50 mg/L hygromycin. After 4 weeks, resistant calli will be transferred to shoot medium to induce shoot growth. The regenerated shoots will be further grown and moved to root regeneration medium to obtain full T0 plants. The transgenic plants will be verified by PCR. For each vector, at least 30 T0 transgenic plants will be used for evaluating the base editing efficiency and OsBBM1 activation activity. We expect that CBE-TALE-Combo constructs will generate higher base editing efficiency at the plastid target genes (PsaA and PsbA). In a separate experiment, we will regenerate rice in the absence of plant hormones in the medium. Under this condition, we only expect to recover transgenic plants based on the CBE-TALE-Combo constructs because the activation of OsBBM1 in callus enables hormone-free plant regeneration. Importantly, we expect to achieve homozygous editing (editing of all plastid genomes regardless of the copy number) due to the enrichment of genome editing using this hormone-free method, as we found with CRISPR-Combo. In the next generation, we expect to obtain chloroplast base-edited plants that segregate out the TALE-Combo transgene.Objective 3: Develop an efficient ABE-TALE-Combo system to enrich the homoplasmic A-to-G editing in rice.Similar to the cloning procedures described in Objective 2, the gBlock gene fragments encoding chloroplast target peptide with a triple FLAG-tag (Left TALE) or a triple HA-tag (Right TALE), rice-codon optimized split-DddAtox and adenine deaminase (AD) will be synthesized by IDT with both KpnI and AatII restriction enzyme sites. These gBlocks will be cloned into pYPQ121 between KpnI and AatII sites, separately, which can generate pYPQ121-2 (that contains cpDdABE). The Left and Right TALE repeats targeting PsaA and PsbA will be excised from pZHY500 and ligated into pYPQ121-2 between XbaI and BamHI sites or NheI and BglII sites, respectively. The final T-DNA vectors will be made through Multisite Gateway reaction with pYPQ121-2, pYPQ127B (the same OsBBM1 activation construct used in Objective 2) and the destination vector pYPQ203. The resulting two ABE-TALE-Combo T-DNA vectors will be used to transform rice protoplasts to test the efficiency of A-to-G base editing in plastids and OsBBM1 activation. Once these vectors show both editing and activation activity in the rice protoplasts, they will be transformed into rice calli. Following the same procedures in Objective 2, both hormone-containing (standard transformation condition) and hormone-free medium will be used for transgenic plant regeneration during tissue culture. It is expected that ABE-TALE-Combo systems will be highly efficient in generating homoplasmic editing lines. In the next generation, transgene-free plants with A-to-G base editing in chloroplast genomes will be obtained.