CHIMERIC CAS9 AND CPF1 PROTEINS FOR TRANSCRIPTION REGULATION AND PROMOTER MODIFICATION IN PEANUT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1015102
• Grant No.: 2018-67014-27572
• Project No.: ALX-GH2017-1
• Proposal No.: 2017-07732
• Program Code: A1141
• Project Start Date: Feb 15, 2018
• Project End Date: Feb 14, 2023
• Grant Year: 2018

Project Director
He, G.

Recipient Organization
TUSKEGEE UNIVERSITY
(N/A)
TUSKEGEE,AL 36088

Performing Department
Col. of Ag. Env. and Nutr. Sci

Non Technical Summary
Peanut breeding has contributed greatly to production, quality and nutritional improvement through dynamic and continual varietal enhancement, which positively impacts the peanut industry. However, conventional breeding is a time-consuming approach, normally taking 10 - 12 years to produce new varieties. In addition, traditional breeding is now constrained by a decline in existing genetic variation. Gene editing is a method used to make any targeted genetic change to a chromosome that produces a viable organism. For this proposal we will assess the suitability of gene-editing via CRISPR-based transcription regulation and cytidine deaminase-based C to T modification in peanut. The methodology we will use in this project is similar with electrical swatch to be on or off on gene expression, rather than directly knockout the genes like cutoff electrical wire. The reliable protocols would be used on other genes of interest.

Animal Health Component

0%

Research Effort Categories

Basic

80%

Applied

(N/A)

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1830	1081	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1830 - Peanut;

Field Of Science
1081 - Breeding;

Keywords

cas9

cpf1

peanut

promoter

transcription regulation

Goals / Objectives
The goal of this proposed project is to use genomic tools to breed for improved peanut varieties through CRISPR-based RNA-guided transcription regulation techniques and promoter sequence modification. Genome-editing via CRISPR technology is the most promising of all known techniques owing to ease of use, high efficiency, success in a wide range of species and variety of application to include multiple genome-editing and gene regulation methods. Because CRISPR technology remains an unknown in peanut, we will select the well-characterized fatty acid desaturase (FAD2) genes as a model due to their simplicity, known mutant effects, trait value and scientific interest. The specific objectives will be 1) Design and validate multiple CRISPR sgRNAs for FAD2 promoters using yeast and hairy root assays; 2) Generation of peanut expressing CRISPR repressors to demonstrate FAD2 gene expression modulation of oleic acid accumulation; 3) Generation of peanut expressing CRISPR cytidine deaminase to demonstration FAD2 promoter modification that lowers gene expression for oleic acid accumulation.

Project Methods
1. Design and validation of multiple CRISPR sgRNAs for use against FAD2 promoters using yeast and hairy root assays.Peanut FAD2 promoters are AT rich having only about 35% GC content. CRISPR targets with low GC content may be less efficient so target validation is vital to ensure success prior to investing in peanut transformation. FAD2 promoters from breeding line 1516-27 will be PCR amplified and sequenced to verify actual DNA sequence prior to starting analysis. In addition, RT-PCR and 5' RACE will be used to verify FAD2 mRNA splice sites and transcription start sites to ensure CRISPR constructs are properly targeted. RT-PCR and 5' RACE will be carried out using embryo, cotyledon, stem, root and leaf tissues. Using existing sequences, we have compared FAD2 promoters from A. duranensis, A. ipaensis (diploid peanut) and A. hypogaea (commercial peanut - allotetraploid) to identify conserved promoter regions. Based on mRNA sequences and homology between species, a 1.7 kb region 5' of the FAD2 coding sequence has been identified as the probable promoter region, which contains about 91 potential CRISPR/Cas9 target sites (averaging one every 20 bp) and 164 CRISPR/Cpf1 target sites (averaging one every 10 bp). The majority of CRISPR targets are shared between the two FAD2 promoter regions likely allowing both genes to be simultaneously silenced using a single construct. In addition, the sequences were scanned with transcription factor binding site prediction software (PlantPAN 2.0). Analysis revealed that two of the three mRNA clusters have a predicted TATA binding protein site just upstream and the third cluster has two TATA box candidates just upstream. Each predicted TATA box is accessible by either Cas9 or Cpf1 or both. The first predicted TATA box is associated with a WRKY and a homeodomain transcription factor (TF) binding site, which has five Cas9 and three Cpf1 targets. The second TATA box is associated with an AP2 TF binding site and has two Cas9 and five Cpf1 target sites. The third TATA box is associated with four myb and two AP2 TF binding sites and has two Cas9 and five Cpf1 targets.We will initially test two potential target sequences for each TATA box using CRISPR/Cas9 and CRISPR/Cpf1 and targets for TF binding sites in yeast and peanut hairy root assays for a total of 21 targets. These sites will be used to block expression using repressor fusions or make C to T changes in TF binding sites in an effort to down regulate FAD2 expression. Gene editing data from these assays will demonstrate that Cas9 and Cpf1 can act upon specific targets in yeast and that they are accessible in peanut root tissue.Yeast is a facile test system for new genome engineering technology. Briefly, CRISPR/Cas9 or CRISPR/Cpf1will be expressed in an "a" mating type cell while a reporter gene that contains a CRISPR target sequence flanked by a small internal duplication that inactivates the reporter will be expressed in an "alpha" mating type cell. After mating, the inactive reporter will be broken at the target site by Cas9 or Cpf1 and the internal duplication healed through homologous recombination, which activates the reporter. Our reporter gene is fluorescent mCherry and its activity can be measured quantitatively. These data will accurately show how well a specific CRISPR/Cas9 or CRISPR/Cpf1 target functions in comparison to experimental controls. Targets that demonstrate a high level of efficiency as compared to controls will be moved on to the peanut hairy root assay.We have established Agrobacterium rhizogenes induced peanut hairy roots as a CRISPR/Cas9 test system and will adapt CRISPR/Cpf1 to the assay as well. This assay is relatively fast and allows the study of gene editing activity in lieu of regenerated plants. Briefly, targets that are validated in the yeast assay will be cloned into our CRISPR/Cas9 high efficiency peanut construct or our developing CRISPR/Cpf1 construct then transformed to A. rhizogenes strain K599. Sequence verified K599 colonies will be used to infect peanut root tissue then incubated at 22o C until hairy roots appear. Hairy root tissues that grow are necessarily transgenic and will be tested for evidence of mutation at the target site using PCR and T7 assays followed by sequence verification of mutations.2. Generation of peanut expressing dCas9-r or dCpf1-r and demonstration of lower FAD2 expression and oleic acid accumulation.We have previously generated a CRISPR/Cas9 construct that reliably produces mutations in 10% of peanut hairy roots. We will make the D10A and H840A mutations in this construct to reproduce dCas9 and the E993A mutation in CRISPR/Cpf1 to produce dCpf1 then add the repressor sequences ELLD or 3x SRDX to the C-terminus as a fusion. The strong constitutive ubiquitin promoter that drives dCas9-r or dCpf1-r is strong enough to ensure that dCas9-r or dCpf1-r are expressed in all target tissues. Our CRISPR system is capable of simultaneously expressing up to four sgRNAs. ELLD and 3xSRDX dCas9-r or dCpf1-r constructs expressing combinations of the most efficient sgRNAs for each independent TATA box domain will be generated: CRISPR/Cas9 and CRISPR/Cpf1 will each have one construct for each TATA box domain, one construct designed to target all three TATA boxes and three constructs designed to target TATA box pairs (1+2, 2+3 and 1+3) with up to four sgRNA's each for a total of fourteen constructs.We will carry out cotyledonary node transformation using breeding line 1516-27 for transformation because of its high transformation efficiency compared to other genotypes, however, other genotypes are available if required. The constructed vectors will be mobilized into Agrobacterium tumefaciens strain EHA105. The cotyledonary nodes from 6-8 days old seedling will be used as explants, and immersed in the infection medium for 1 h with gentle shaking. Co-cultivation will be for 3 days at 26±1o C in the dark. After co-cultivation, cotyledonary nodes will be transferred to SIM2 medium containing 300 mg/L cefotaxime and 300 mg/L timetin at 28o C under a photoperiod of 16 h/8h (light/dark) for 2-3 weeks. Calli producing shoot-like structures will be transferred to RM (MS salts and vitamins, 3% (w/v) sucrose, 5.37 uM NAA, and 0.8% (w/v) agar) for root induction. Once roots are generated, plantlets will be transplanted in pots at 28o C under a photoperiod of 16 h/8 h.Putative transformants will be regenerated and screened at the plantlet, maturing tissue and seed set stages using RT-PCR to detect Cas9 or Cpf1 and FAD2 transcripts in embryo, cotyledon, stem, root and leaf tissues followed by q-PCR using the same tissues for plants that appear to have altered FAD2 expression patterns. Seeds from fully regenerated plants that express dCas9-r or dCpf1-r will be used for fatty acid analysis by gas chromatography.3. Generation of peanut expressing nCas9-cd or dCpf1-cd and demonstration of lower FAD2 expression and oleic acid accumulationWe will make a specific mutations (D10A) in our peanut CRISPR/Cas9 and (E993A) in CRISPR/Cpf1constructs to reproduce nCas9 and dCpf1 then add cytidine deaminase (CD) and uracil glycosylase inhibitor (UGI) fusion sequences to generate nCas9-cd and dCpf1-cd. Two construct types will be generated: 1) peanut codon optimized PmCDA1 encoding the sea lamprey CD gene will be fused to the N-terminus of nCas9 and dCpf1 and the UGI sequence will be fused to the C-terminus of each; 2) peanut codon optimized APOBEC1 encoding the rat CD gene will be fused to the N-terminus of nCas9 and dCpf1, and the UGI sequence will be fused to the C-terminus of each. Two PmCDA1 and two rat CD CRISPR/nCas9 constructs with identical targets will be generated and transformed to peanut hairy roots then mutation frequency will be measured by PCR and sequencing to determine which is more efficient. The same experiments will be performed with the dCpf1-cd construct versions.

Progress 02/15/18 to 02/14/23

Outputs
Target Audience:Plant scientists and peanut breeders Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has been provided opportunities for training graduate students in genome editing technology. How have the results been disseminated to communities of interest?We have published several articles in peer-review journals and presented our results in scientific meeting. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? To exploit potential CRISPR/Cas-mediated genome editing technology for developing peanut plants with improved traits of interest, we selected the fatty acid desaturase 2 (FAD2) genes as targets because of their simplicity, known mutant effects, trait value and scientific interest. We then designed constructs for different needs in peanut genome editing applications, including knock-out, base editing, and regulatory editor for manipulation of gene expression. A total of 15 constructs have been developed with different components of the CRISPR/Cas system that are potentially useful in peanut genome editing in our laboratory. For these constructs, the guide portion of the sgRNA is inserted as phosphorylated and annealed primer pairs designed with single stranded end sequences that match the BsaI digested vector ends. Guide RNAs specific to a target in the coding region and the predicted RY and 2S seed protein motif CREs in the promoter of FAD2 genes were designed using the Cas-Designer tool.Oligonucleotide pairs used for guide RNAs were synthesized at the Iowa State University DNA facility and were annealed after phosphorylation to generate sticky ends that correspond to the overhangs generated by BsaI restriction digestion of the expression vectors to complete the sgRNAs of each construct. In addition to using one sgRNA to target single site, we also used multiplex sgRNAs to target both RY and 2S motifs simultaneously. The cloning strategy for dual sgRNA constructs was based on a one step Golden Gate ligation process. The CRISPR/Cpf1 system possesses unique features such as a single shorter crRNA that cleaves DNA without an additional trans-activating crRNA (tracrRNA) as used in Cas9, targets T-rich regions of the genome, cleaves distal to the PAM, and generates staggered ends when cleaving target sites. We have generated two constructs with two Cpf1 orthologs, LbCpf1andAsCpf1, respectively, to determine the effect of different Cpf1 variants on editing efficiency in peanut. Moreover, to test potential regulation of gene expression in peanut, the repressor 3xSRDX was fused to dCas9 with the extended scaffold or standard scaffold to generate constructs or fused to dLbCpf1. These three constructs target the FAD2 gene promoters to test repression of gene expression. Similarly, the TV activator was fused to dCas9 with the extended scaffold or standard scaffold to generate constructs or dLbCpf1 producing a construct and used to test activation of gene expression. To induce point mutations in the coding region or promoter sequence of FAD2 genes, the sea lamprey CDA (PmCDA1) or rat CDA (rAPOBEC1) coding sequences were fused to nCas9 with different sgRNA scaffolds, respectively. Rat CDA was also fused to dLbCpf1 to generate a construct. Using our developed constructs with different sgRNA scaffolds, more than 90% of detected mutations were deletions ranging from one nucleotide to 57 nucleotides in the coding region of both FAD2 homoeoalleles. Although these constructs are suitable for gene editing in peanut, the editing efficiency for the construct using the extended scaffold was 32%, but only 24% for the construct using the typical scaffold. These constructs were also used to disrupt the combination of cis-regulatory elements in the FAD2 promoters that are thought to be bound by transcription factors. The RY repeat element (CATGCATG) and 2S seed protein motif (CAAACAC) are two crucial cis-regulatory elements for regulating expression activity in a number of seed-specific promoters. The objective of knocking out the RY and 2S elements in the 5' UTR of FAD2 genes was for functional characterization of these elements and to potentially generate seeds with increased oleic acid content without affecting the fatty acid composition in other plant tissues. The single sgRNA constructs targeting the distal RY motif resulted in indels from +2 to -33 bp and the standard sgRNA scaffold performed better than the extended gRNA scaffold. The number of mutations detected at the proximal 2S motif were lower than that observed at the RY element and, in contrast, the extended scaffold performed better than the standard sgRNA scaffold. When dual sgRNAs were used to target RY and 2S simultaneously, the use of two gRNAs in the construct had little effect on the editing efficiency. Overall editing efficiency at RY was greater than at 2S regardless of sgRNA order in the constructs, which was similar to the results of the single sgRNA studies. However, the duel sgRNA construct did generate a single instance of a large 1,140 bp deletion between the RY and 2S motifs. CRISPR/Cpf1-mediated genome editing has been widely utilized in plants due to its versatility and simplified features. Two constructs with different Cpf1 orthologs, AsCpf1 and LbCpf1, were tested to demonstrated genome editing activity in peanut. Our preliminary data indicated that the construct/LbCpf1 performed with higher editing activity compared to the construct/AsCpf1. Agrobacterium-medicated transformation is a popular system for delivering genes into peanut cells. Despite significant improvement in transformation protocols have been achieved and genes of interest were delivered into the peanut genome via Agrobacterium-medicated transformation, only a few peanut genotypes are known to have a high transformation efficiency. Among these parameters, the major limitation in peanut transformation is the tissue culture procedure for regeneration of adventitious shoots from explants, which is time-consuming, recalcitrant to regeneration and genotype-dependent. Developing an alternative that breaks the tissue culture bottleneck in the transformation system would be considered as advantageous. To exploit the potential of CRISPR/Cas components delivery into peanut cells, we used an in planta transformation method, called calyx tube (hypanthium) injection-based transformation. Previously validated constructs were used to target the coding region and RY and 2S motifs in the promoter of FAD2 genes, and were individually mobilized into Agrobacterium tumefaciens strain GV3101. Agrobacterium resuspensions were injected into the middle of the calyx tube with an insulin syringe between 9-10 am every morning during the flower blossoming season. A total of 2,521 peanut flowers from three genotypes were injected with Agrobacterium and 363 T0 seeds were harvested. Results showed that 28.9% (105/363) of harvested seeds showed increased oleic acid content, indicating that these 105 seeds contained FAD2 mutations although the rate of oleic acid increase in the three genotypes was different. Among edited seeds, 85.7% (90/105) of seeds showed a slight increase in oleic acid content between 55-60%, 12.4% (13/105) showed a significant increase of 61-65%, and 1.9% (2/105) showed a very significant increase in the level of oleic acid at 66-70%. None of the seeds reached 80% oleic acid levels as seen in naturally occurring mutants. However, amplifications of T1 plant DNAs showed no mutations in the targets of the FAD2 genes, and all seeds harvested from T1 plants derived from these T0 edited seeds showed the same oleic acid content as wild types. This result might indicate that the CRISPR components were delivered into somatic cells rather than germ cells in T0 seeds using the calyx tube injection method.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Anjanasree K. Neelakandan, Binita Subedi, Sy M. Traore, Papias Binagwa, David A. Wright, Guohao He. (2022) Base editing in peanut using CRISPR/nCas9. Frontiers in Genome Editing, 4:901444.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Anjanasree K. Neelakandan, David A. Wright, Sy M. Traore, Xingli Ma, Binita Subedi, Suman Veeramasu, Martin H. Spalding, Guohao He. (2022) Application of CRISPR/Cas9 system for efficient gene editing in peanut. Plants, 11:1361.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Naveen Puppala, Spurthi N. Nayak, Alvaro Sanz-Saez, Charles Chen, Mura J. Devi, Nivedita Nivedita, Yin Bao, Guohao He, Sy M. Traore, David A. Wright, Manish K. Pandey, Vinay Sharma. (2023) Sustaining yield and nutritional quality of peanuts in harsh environments: Physiological and molecular basis of drought and heat stress tolerance. Frontiers in Genetics, 14:1121462.

Progress 02/15/21 to 02/14/22

Outputs
Target Audience:Scientists in plant research and peanut breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has been provided opportunities for traning graduate students in genome editng technology. How have the results been disseminated to communities of interest?We have published several articles in peer-review journals andpresented our results in scientific meeting. What do you plan to do during the next reporting period to accomplish the goals?In the next period, we will concentrate our experiment on optimizing the protocol of calyx tube injection transformation to obtain edited plants.

Impacts
What was accomplished under these goals? In this period, we utilized CRISPR/Cas9-mediated gene editing to modify cis-regulatory elements in the 5'UTR and intron of FAD2 genes for functional characterization of these elements and to potentially generate seeds with increased oleic acid content without affecting the fatty acid composition in other plant tissues. The RY repeat element and 2S seed protein motif were chosen as targets due to their crucial for activity in a number of seed-specific promoters. We also used two expression vectors as tools to induce point mutations in the promoter and the coding sequences of FAD2 genes. In these two constructs, a single nuclease null variant, nCas9 D10A, was fused to different deaminases: PmCDA1 cytosine deaminase and rAPOBEC1 deaminase with an uracil glycosylase inhibitor (UGI). Three gRNAs were cloned independently into both constructs and the functionality and efficiency were tested at three target sites in the AhFAD2 genes. Both constructs displayed base editing activity in which cytosine was replaced by thymine or other bases in the targeted editing window. Testing of constructs in a hairy root assay demonstrated that the editing efficiency at the distal RY motif was higher than at the proximal 2S motif, and the predominance of edits involving deletions less than 10 bp with a single gRNA target. Taken together, these data demonstrate the efficacy of CRISPR/Cas9 mediated editing of peanut FAD2 promoter sequences and suggest that some positions within a promoter may be more accessible than others, use of different gRNA scaffolds may give some flexibility in target efficiency and there may be some difference in homeolog accessibility when targeting gene families. The PmCDA1 deaminase showed higher efficiency compared to rAPOBEC1, suggesting that the former is a better base editor in peanut. Results showed the increased oleic acid content in T0 edited seeds using calyx tube injection transformation in our last report. However, when tested T1 plant D NAs and T1 seed oleic acid content, we did not find mutations in target DNAs and no increased oleic acid content in T1 seeds. The higher oleic acid content in T0 seeds may be resulted from somatic cell mutation. A further testing will need to optimize the parameter for inoculation media, the suitable time and the location of the calyx tube for injection to improve the delivery of constructs in the calyx tube injection.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Sy Traore, Suoyi Han, Papias Binagwa, Wen Xu, Xiangyu Chen, Fengzhen Liu, Guohao He (2021) Genome-wide identification of mlo genes in the cultivated peanut (Arachis hypogaea L.). Euphytica, 217:61.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Benhui Wei, Suoyi Han, Guohao He (2021) Smash-ridging cultivation improves crop production. Outlook of Agriculture, 1-5.
• Type: Book Chapters Status: Published Year Published: 2021 Citation: Guohao He, Sy M. Traore, Papias H. Binagwa, Conrad Bonsi, Channapatna S. Prakash (2021) Date palm quantitative trait loci. Book Chapter in: Jameel M. AI-Khayri et al. (eds.), The Date Palm Genome, Vol. 2: Omics and Molecular Breeding. https://doi.org/10.1007/978-3-030-73750-4, � Springer Nature, Switzerland.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Papias H. Binagwa, Sy M. Traore, Marceline Egnin, Gregory C. Bernard, Inocent Ritte, Desmond Mortley, Kelvin Kamfwa, Guohao He, Conrad Bonsi (2021) Genome-wide identification of powdery mildew resistance in common bean (Phaseolus vulgaris L.). Frontiers in Genetics, 12:673069.
• Type: Book Chapters Status: Published Year Published: 2021 Citation: Sy Traore, Guohao He (2021) Soybean as a model crop to study plant oil genes: mutations in FAD2 gene family. Book Chapter in: Model Organisms in Plant Genetics. DOI: http://dx.doi.org/10.5772/intechopen.99752.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Anjanasree K. Neelakandan, David A. Wright, Sy M. Traore, Xiangyu Chen, Martin H. Spalding, Guohao He (2022) CRISPR/Cas9 based site-specific modification of FAD2 cis-regulatory motifs in peanut (Arachis hypogaea L.). Frontiers in Genetics, 13:849961. Doi:10.3389/fgene.2022.849961.
• Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Anjanasree K. Neelakandan, Binita Subedi, Sy M. Traore, Papias Binagwa, David A. Wright, Guohao He (2022) Base editing in peanut using CRISPR/nCas9. Frontiers in Genome Editing, (accepted)

Progress 02/15/20 to 02/14/21

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students and one postdoc have been trained in gene editing project. How have the results been disseminated to communities of interest?All results will be submit to scientific journals and present in scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?We will sequence DNAs extracted from the edited seeds and find out the relationship between DNA mutations and the oleic acid level. We will optimize calyx tube injection, such as injection time and position, and continue to verified the rest of constructs we made. For editied T0 seeds, we will conduct genetic study of mutations in the following generations. Several manuscripts will be prepared for publishing.

Impacts
What was accomplished under these goals? In this period, our research activities were focused on comparison of transformation efficiency using the verified CRISPR/Cas9 and Cpf1 constructs with three peanut genotypes. To avoid tissue culture and regeneration in Agrobacterium-mediate transformation that is recalcitrant in peanut, we have used calyx tube injection method. Results showed 105 out of 365 transformed seeds increase the content of oleic acid. Among edited seeds, 85.7% seeds increased oleic acid to 55-60%, 12.4% fell in 61-65%, and 2% assigned to 66-70%, compared to wild types with less than 55% of oleic acid level. Gene editing targeted on promoter has resulted the higher oleic acid level than that targeted on the coding region and the highest oleic acid level was resulted from the mutations on the RY element. Although transformation efficiency is related with the genotype used, the benefit of calyx tube injection is that T0 seeds can be directly obtained as the plant ripen. Compared to the floral dip method, calyx tube injection increased the transformation efficiency, which is a promising way for Agrobacterium-mediated transformation in peanut.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Xingli Ma et al. (2020) Genome-wide identification and analysis of long noncoding RNAs (lncRNAs) during seed development in peanut (Arachis hypogaea L.). BMC Plant Biology 29:192.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Limin Gong, Suoyi Han, Mei Yuan, Xingli Ma, Austin Hagan, and Guohao He (2020) Transcriptomic analyses reveal the expression and regulation of genes associated with resistance to early leaf spot. BMC Research Notes, 13:381. in peanut

Progress 02/15/19 to 02/14/20

Outputs
Target Audience:Plant gene editing research community and peanut breeding community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students and postdoc involved this project have been providedhands-on experiences in plant gene editing and have opportunities to present their results in the professional conferences. How have the results been disseminated to communities of interest?All results obtained from this project have been reported to research communities via APRES annual meeting, National Association of Plant Breeders annual meeting, and Plant and Animal Genome meeting. What do you plan to do during the next reporting period to accomplish the goals?We will continue to test remaining CRISPR constructs we designed. We have started to perform transformation to verify phenotypes with high level ofoleic acid in seeds using those positive constructs. Transformation in peanut is a challenge due to the difficulty in conventional tissue culture-based regeneration processes. We will plan to use the biolistic particle delivery system to shoot meristems for transformation and regeneration.

Impacts
What was accomplished under these goals? We have designed CRISPR/Cas9 and /Cpf1 constructs to edit not only in the CDS region, but also in the promoter region of FAD2 genes. Constructs with Cpf1 and deaminase were used for targeting the CDS region, the functionality of these constructs was validated in peanut through protoplast transfection, leaf infiltration, and hairy root transformation. Our results showed various FAD2 edits via deletion, insertion and cysteine conversion to thymine and the editing efficiency in the CDS was ~ 24%. The constructs with Cas9 were used to target the distal (RY repeat motif) and proximal (2S seed protein motif) regions of the FAD2 promoter. Because RY element and 2S seed protein motifs are implicated in the regulation of seed specific gene expression and linoleic acid may be required for healthy plant growth, gene editing the FAD2 promoter may generate seed with reduced linoleic acid while maintaining normal fatty acid profiles in other plant tissues. Our results showed that indel mutations were generated in the promoter regions and the mutagenesis rate was higher at the distal region (44%) than at the proximal region (9%).

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Mei Yuan, Jun Zhu, Limin Gong, Liangqiong He, Crystal Lee, Suoyi Han, Charles Chen, Guohao He(2019)Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing. BMC Biotechnology 19:24.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Weijian Zhuang et al. (2019) The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication. Nature Genetics, https://doi.org/10.1038/s41588-019-0402-2
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Dongmei Yin et al. (2019) Comparison of Arachis monticola with diploid and cultivated tetraploid genomes reveals asymmetric subgenome evolution and improvement of peanut. Advanced Science, DOI: 10.1002/advs.201901672

Progress 02/15/18 to 02/14/19

Outputs
Target Audience:Plantgene editingresearch community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project have provided opportunities for two graduate students and two postdoc training in gene editing technology. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We are testing our designed 15 CRISPR constructs for the efficency of mutagensis on the FAD2 genes and select the best constructs for gene editing.

Impacts
What was accomplished under these goals? We have designed 15 CRISPR constructs. Among these constructs, there are four principal nuclease constructs for gene disruption, three with repressors (3xSRDX) and three with activators (TV) that will be used in the promoter to manipulate gene expression, and five constructs for base editors - one set uses sea lamprey CDA and the other set uses a rat CDA. The last five constructs for base editing should be used in either the promoter or the coding sequence. These constructs were designed to have a guide RNA inserted at the two BsaI sites in a single cloning step with both spectinomycin resistant at 100 micrograms/ml and kanamycin resistant to at least 30 micrograms/ml. Each of 15 constructs was added a gRNA that induced mutations in our previous study and has been testing their efficiency of mutagenesis.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Mei Yuan, Jun Zhu1, Limin Gong, Liangqiong He, Crystal Lee, Suoyi Han, Charles Chen, Guohao He. (2019)Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing. BMC Biotechnology (under review)