Source: TUSKEGEE UNIVERSITY submitted to
GENOME EDITING OF FAD2 GENES TO IMPROVE OIL QUALITY IN PEANUT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1015103
Grant No.
2018-38821-27758
Project No.
ALX-GH2017-2
Proposal No.
2017-07405
Multistate No.
(N/A)
Program Code
EQ
Project Start Date
Mar 1, 2018
Project End Date
Feb 28, 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 (Arachis hypogaea L.) is one of the major oil and food crops worldwide. It is cultivated in more than 100 countries. All parts of the crop are useful but the most important part is the seed which is used for oil extraction. A good oil quality in peanut seeds is important to meet the needs of peanut farmer, industry processors, and consumers. The flavor and quality of either seed or oil is dependent on the fatty acid composition of extracted oil. Oils containing high percentage of linoleic acid are prone to oxidation, leading to rancidity, off flavors, and short shelf-life. Oleic acid has 10-fold higher auto-oxidative stability than linoleic acid; therefore, high O/L peanut has a longer shelf life. Oils with high levels of oleic acid are also nutritionally beneficial. Therefore, efforts to increase oleic acid content in peanut seeds will provide a better quality and healthier oil for consumers. Precise modification of the target genes in a given genome by the CRISPR/Cas system provides an excellent tool to validate specific gene function or modify known genes. Through gene-editing, we expect to generate mutations in the FAD2 genes that result in diminished function, thus increasing the level of oleic acid in peanut seeds. The results of this proposed project will provide an alternative source of high oleate materials for peanut breeding, in turn, supplying high quality peanut oil for consumers.
Animal Health Component
0%
Research Effort Categories
Basic
70%
Applied
(N/A)
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20118301040100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1830 - Peanut;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The goal of this proposed project is to improve peanut oil by gene-editing FAD2 genes through CRISPR/Cas9 technology. The FAD2 genes encode the enzyme fatty acid desaturase that catalyzes the conversion of oleic acid to linoleic acid. Because oleic acid has a high level of auto-oxidative stability, peanut seeds with a high level of oleic acid have a longer shelf life and are nutritional beneficial for consumers. Modification or disruption of FAD2 genes will reduce the activity of this enzyme to convert oleic acid to linoleic acid, thus to accumulate the level of oleic acid in seeds. The specific objectives will be 1) Design of multiple sgRNAs for different target regions in the FAD2 genes and development of suitable CRISPR constructs; 2) Validation of CRISPR DNA constructs and ribonucleoprotein (RNPs) complexesthrough mutations generated by protoplast transfection or hairy root transformation; 3) Cotyledonary node transformation with positive constructs and development of regeneration system.
Project Methods
1. Design of multiple sgRNAs for different target regions in the FAD2 genes and development of suitable CRISPR constructsSince there are no reports of genome editing in peanut, we must first establish functional gene-editing in this crop and optimize the needed vectors. The sequences of FAD2 genes will be used to identify suitable target sites and design sgRNAs to simultaneously target several specific regions within the genes or target separate homeologs with multiple sgRNAs. Three strategies will be used to accomplish the latter: 1) mutant U6 promoters will be used to drive sgRNA expression, which may lower targeting efficacy and generate heterozygotes in either homeolog set based on experience with maize; 2) use of sgRNAs that have mismatches in the seed section of the sgRNA to reduce targeting efficiency to produce heterozygotes in either homeolog set based on experience in rice; and 3) make use of base differences between homeologs to generate sgRNAs that may target one homeolog, but not the other. Other criteria for sgRNAs design will consider (i) the NGG-3' PAM for Cas9 and (ii) minimization of off-target activity through use of the CRISPR Genome Analysis Tool (http://cbc.gdcb.iastate.edu/cgat/). Designed sgRNA will be inserted between the MtU6 promoter and gRNA scaffold from the pUC gRNA Shuttle plasmid (Addgene plasmid 47024). Constructs will be developed by inserting the sgRNA target cassette into the p201B:Cas9 vector. In addition to CRISPR/Cas9, we will also test CRISPR/Cpf1 for efficacy in peanut. Unlike Cas9, Cpf1 creates DSBs with a 5' over hang, it has a T rich 5' PAM and uses a much shorter guide RNA. Because Cpf1 recognizes T-rich 5' PAM, we will design gRNAs for Cpf1 based on Cas-Designer (http://rgenome.ibs.re.kr). The Cpf1 coding region of each plasmid will be substituted for Cas9 in the above vector sets and suitable gRNAs will be inserted for expression.In an effort to better control CRISPR/Cas9 and CRISPR/Cpf1 activity, RNP complexes for each nuclease will be generated from commercially available protein and RNA sources such as Integrated DNA Technologies (IDT) or New England Biolabs (NEB). RNPs along with a selectable marker will be electroporated or transfected into peanut protoplasts to test their efficacy against the FAD2 genes. In addition, use of Cpf1 RNP may reduce off-target effects and efficiently induce insertion/deletion (indel).2. Validation of CRISPR DNA constructs and RNPs through mutations generated by protoplast transfection or hairy root transformationTo initially evaluate DNA and RNP delivery and performance in vivo, an established peanut protoplast culture method and hairy root culture will be employed. Briefly, expanded young leaves from 2-3 week old peanut plants will be cut into 0.5 - 1 mm leaf strips that will be digested with an enzyme solution and protoplasts will be spun down and resuspended in MMg (0.4 M mannitol, 15 mM MgCl2, and 4 mM MES-K) solution before PEG transfection. Constructed CRISPR plasmids or preassembled LbCpf1 and AsCpf1 RNPs will be delivered into protoplasts by PEG treatment. After 24 hours' incubation at 22o C, genomic DNA will be isolated from protoplasts then FAD2 gene sequences will be PCR amplified. PCR products will be analyzed using Illumina deep sequencing.Plant regeneration from peanut protoplasts is currently not possible and detailed methods for CRISPR construct DNA and RNP delivery to intact peanut tissue is not completely understood. To better understand these details, we will deliver DNA and RNPs that pass the protoplast test to peanut root tissues. DNA delivery to root tissues will be accomplished by Agrobacterium rhizogenes strain K599 mediated transformation using selectable marker bearing T-vector CRISPR plasmids. Transgenic hairy roots are necessarily generated by Agrobacterium infection and selection ensures that all hairy roots contain the CRISPR construct. Root tissues will be incubated at 22o C on selective media until hairy roots appear then FAD2 target analysis will be performed. Biolistic RNP delivery will occur in combination with a selectable marker using root tissues previously infected by Agrobacterium strain K599 without a T-vector. Analysis of FAD2 targets from genomic DNA will proceed in a similar manner as with protoplasts. Such in vivo prescreening of CRISPR DNA and RNP complexes enables us to identify highly active CRISPR DNA constructs or RNPs and their delivery conditions prior to attempts at regenerating peanut plants containing FAD2 mutations.3. Cotyledonary node transformation with positive constructs and development of regeneration systemOnce highly active CRISPR DNA constructs or RNPs and delivery conditions are identified, we will carry out cotyledonary node transformation. The selected genotype will be transformed with optimized target sequences. Briefly, seeds will be sterilized by immersion in 75% (v/v) ethanol for 30 sec, then in 4% (w/v) sodium hypochlorite for 30 min, and followed by washing for 4-6 times with sterile water. The constructed vectors will be mobilized into Agrobacterium tumefaciens strain EHA105. A single bacterial colony will be inoculated into 50 ml of liquid YEB medium containing 50 mg/L kanamycin and incubate at 28o C and 200 rpm overnight. Bacterial cells at OD600=0.5 will be collected and resuspended in the liquid AB infection medium (5 g sucrose, 25 g of D-sorbitol, 50 ml of AB salts) with 50 mg/ml kanamycin. The cotyledonary nodes from 6-8 days old seedling will be used as explants for both Agrobacterium-mediated transformation and biolistic RNP delivery. For Agrobacterium-mediated transformation, cotyledonary nodes will be 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.Genomic DNAs will be isolated from putatively edited lines at the callus stage and at the plantlet stage, followed by sequencing of PCR amplicons to verify edits; at the callus stage for evidence of editing, and putatively edited transformants will be regenerated and rescreened at the plantlet stage. Seeds from fully regenerated plants with verified target gene disruptions via CRISPR technology will be used for fatty acid analysis by gas chromatography. Fatty acid composition from these mutant lines will allow us to understand the relationships between different FAD2 genes and their alleles as well as between genes and phenotypes. Genetic analysis of progeny will demonstrate heritability of FAD2 mutations.

Progress 03/01/18 to 02/28/23

Outputs
Target Audience:Plant biotechnology and peanut research community. 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 and postdoctors 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. Design considerations included seed motif regions which are 8-12 nucleotides proximal to the PAM sequence for Cas9 nucleases and 5-6 nucleotides for Cpf1 nucleases while regarding more distal sequences to be less important for specificity. This was due to sequence differences between the promoters in the two FAD2 genes. In addition, a 17-bp truncated guide RNA or a longer guide RNA with two additional guanidine residues at the 5' end was designed to potentially reduce off-target issues. 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. 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. To establish an efficient base editing system in peanut, two constructs with nCas9 fused to different deaminases were tested as tools to induce point mutations in the promoter and the coding sequences of peanut FAD2 genes. Both constructs harbor the single nuclease null variant, nCas9 D10A, to which the PmCDA1 cytosine deaminase was fused to the C-terminal while rAPOBEC1 deaminase and an uracil glycosylase inhibitor (UGI) were fused to the N-terminal and the C-terminal respectively. Three gRNAs were cloned independently into both constructs and the functionality and efficiency were tested at three target sites, RY and 2S in the promoter and one site in the coding region of FAD2 genes. Both constructs displayed base editing activity in which cytosine was replaced by thymine or other bases in the targeted editing window. The construct containing PmCDA1 showed higher efficiency compared to the construct with rAPOBEC1 suggesting that the former is a better base editor in peanut. 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 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, 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: 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, 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.
  • 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 03/01/21 to 02/28/22

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 provided training opportunitiesfor two graduate studends, who were involved in CRISPR/Cas9-based gene editing. How have the results been disseminated to communities of interest?Our results derived from the project have been reported in peer-review journals and will be presented in scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?In the next period, we will focus our research activity on the optimization of calyx tube injection method.

Impacts
What was accomplished under these goals? In this period, we have 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 selectedas targets due to their crucial for activity in a number of seed-specific promoters. 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. 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 DNAs 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: 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.). Front Genet, 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: Under Review Year Published: 2021 Citation: Anjanasree K. Neelakandan, David Wright, Sy M. Traore, Xingli Ma, Binita Subedi, Suman Veeramasu, Martin H. Spalding, Guohao He (2021) CRISPR/Cas9 toolbox for targeting gene modification in peanut. Plant Biotechnology Report.
  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Anjanasree K. Neelakandan, David A. Wright, Sy M. Traore, Xiangyu Chen, Martin H. Spalding, Guohao He (2021) CRISPR/Cas9 based site-specific modification of FAD2 cis-regulatory motifs in peanut (Arachis hypogaea L). Frontiers in Genetics.


Progress 03/01/20 to 02/28/21

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 those DNA samplesextracted from the edited seeds and determine how DNA sequence changes affect the level of oleic acid in peanut seeds. We will optimize calyx tube injection method, such as what is the best time and position on calyx tube for injection. Also, we will continue to verified the rest of constructs we made. For edited T0 seeds, we will conduct genetic study of mutations in the following segregation generations. Several manuscripts have beenpreparingfor publication.

Impacts
What was accomplished under these goals? In this period, we have tested constructs with deaminase for base editing and those with LbCpf1 and AsCpf1 for gene editing on FAD2 genes. The resultshowed these constructs did successfully induce mutations in the coding region of FAD2. We also performed the comparison of transformation efficiency using these verified constructs via Agrobacterium-mediate transformation in three peanut genotypes. To avoid tissue culture and regeneration that are not only time-consuming but also 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 the level of 55% oleic acid. 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 on the promoter. Although transformation efficiency depended onthe 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 03/01/19 to 02/29/20

Outputs
Target Audience:Research community in genome editing and peanut breeding. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students and postdoc involved this project have been provided hands-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 have started to perform transformation to verify phenotypes with high level of oleic acid in seeds using those positive constructs. Transformation in peanut is a challenge due to the difficulty in conventional tissue culture-based regeneration processes. Weare trying the transformation byflower dipping and also plan to use the biolistic particle delivery system to shoot meristems or calluses for transformation and regeneration.

Impacts
What was accomplished under these goals? In our previous report, we tested a soybean CRISPR/Cas9 constructwith ourdesigned gRNA targeting on the peanut FAD2 genes. The results showed thatinduced mutations were same as natural mutations in this gene. In this period, we have designed several CRISPR/Cas9 and Cpf1 constructs and several gRNAs targeting on different locations in the coding region of FAD2. Many mutations including deletion, insertion and point mutations have been sucessfully induced using these editing tools. The editing efficiency was 23-28%. Among these edited sequences, ~38% were monoallelic and only 0.8-15% mutations induced in both alleles whenusing different constructs. It seems the editing efficiency in FAD2B was slightly higher than in FAD2A.

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 03/01/18 to 02/28/19

Outputs
Target Audience:Plant molecular research community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Twograduate students andtwo postdoctoral fellowshave opportunities for training in CRISPR/Cas9 technology andleadership skills. How have the results been disseminated to communities of interest?We will present our results in peanut annual meeting and international peanut genomics conference. What do you plan to do during the next reporting period to accomplish the goals?We will plan to test our improved constructs and select best constructs for gene editing and transformation.

Impacts
What was accomplished under these goals? The hot spot of natural mutation in these genes was selected as the target region. Appropriate sgRNAs were designed and cloned into a CRISPR/Cas9 expression plasmid. As a result of CRISPR/Cas9 activity, three mutations were identified - G448A in ahFAD2A, and 441_442insA and G451T in ahFAD2B. The G448A and 441_442insA mutations are the same as those seen in existing high oleate varieties and the G451T is new mutation. Because natural mutations appear more often in the ahFAD2A gene than in the ahFAD2B gene in subspecies A. hypogaea var. hypogaea, the mutations induced in ahFAD2B by gene editing may be useful in developing high oleate lines with many genetic backgrounds after validation of oleic acid content in the transformed lines. The appearance of the G448A mutation in ahFAD2A is a further benefit for high oleic acid oil content. Fifteen improved constructs weredesigned for gene editing, base editing, and promoter editing purposes. The gRNAshave been added into those constructsthat are testing now.

Publications

  • Type: Journal Articles Status: Submitted 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 (under review)