Source: MICHIGAN STATE UNIV submitted to NRP
GENOME WIDE EVALUATION OF OFF-TARGETS FROM GENE EDITING REAGENTS IN SEED VS. VEGETATIVELY PROPAGATED CROP SPECIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1017103
Grant No.
2018-33522-28736
Cumulative Award Amt.
$500,000.00
Proposal No.
2018-02675
Multistate No.
(N/A)
Project Start Date
Sep 1, 2018
Project End Date
Aug 31, 2023
Grant Year
2018
Program Code
[HX]- Biotechnology Risk Assessment
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
PLANT SOIL MICROBIAL
Non Technical Summary
Due to ease of use, CRISPR-Cas9 has advanced remarkably in the past few years and has become a ubiquitous genome-editing method being applied to an increasing number of species. While several successful cases of crop improvement using various genome- editing platforms including CRISPR systems and TALENs have been reported, concerns of genome wide off-targets have not been thoroughly addressed. Tremendous progress has been made in increasing the specificity and thus reducing off-targeting by engineering Cas9 variants such as the hyper-accurate Cas9 variant, an alternative CRISPR-Cpf1 system analogous to CRISPR-Cas9, and a new base editing approach, which doesn't require DNA cleavage to incorporate specific mutations. Regulatory agencies need to evaluate the precision of these new and emerging gene- editing reagents. We propose to use the current and emerging genome-editing reagents for targeted mutagenesis of commercially important candidate genes in two different crop species, potato and camelina, which have contrasting genomic architecture and modes of reproduction. In potatomechanicaldamage totubers during harvest and post-harvest procedures lead to discoloration due to enzymatic browning caused by the candidate genePotato Polyphenol oxidase (PPO). PPO catalyzes the conversion of o-dihydroxyphenols to o-quinones. The quinones polymerize and react with amino acids resulting in the formation of black/brown pigmentation. Our second candidate gene, delta-12-desaturase (FAD2) converts oleic acid (18:1) fatty acid to 18:2. By knocking outFAD2 we retain high oleic acid which is desirable in industrial oils.We will compare the genome wide off-target effects caused by these reagents using whole genome sequencing and determine the type and frequency of on- and off-target variation attributable by genome-editing regeants. This study would provide a comprehensive risk assessment of genome- edited crops with current technologies. This proposal addresses the BRAG focus area of "Comparison of the types and frequencies of nucleic acid changes introduced into important crop genomes, via genetic insertion/modification techniques versus other plant breeding techniques - Off target effects of genome-editing technologies".
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011310108110%
2011310108020%
2011310104040%
2011899104030%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1310 - Potato; 1899 - Oilseed and oil crops, general/other;

Field Of Science
1040 - Molecular biology; 1081 - Breeding; 1080 - Genetics;
Goals / Objectives
We propose to use CRISPR/Cas9, CRISPR/Cpf1, CRISPR/HypaCas9, TALENs and base editors to generate targeted knock-outs in genes of commercial importance in potato and camelina.Objective 1:Generate 10 independent gene edited lines ofPolyphenol oxidases(StuPPO1-4)in potato with each genome-editing reagent: CRISPR/Cas9, CRISPR/Cpf1, CRISPR/HypaCas9, plant base editors and TALENs. (Year 1)We willGenerate 10 non-transformed regenerant lines and 10 vector-control transformed lines to provide a baseline of mutations induced by tissue culture, regeneration, and transformation.Objective 2:Generate 10 independent gene edited lines ofdelta-12-desaturase (FAD2)in camelina with each genome-editing reagent: CRISPR/Cas9, CRISPR/Cpf1, CRISPR/HypaCas9, plant base editors and TALENs. (Year 1)We willUse single seed descent to generate 10 independent camelina accessions to quantitate inherent rates of mutation. Generate 10 vector-control transformed lines to determine mutation rate associated with transformation.Objective 3:Compare specificity of genome-editing reagents using whole genome resequencing and evaluate genome wide off-target frequencies across reagents and crops. (Years 2 and 3)We will employ whole genome resequencing and two complementary bioinformatics methods to determine the on- and off-target effects of genome editing, tissue culture and regeneration, transformation, and sexual reproduction.
Project Methods
Objective 1:Generate 10 independentPPOknock-out lines in DMRH S5 28-5 potato with five genome-editing reagents.Two target regions will be selected from conserved regions of the potato tuber expressedPPOgenes,StuPPO1 - StuPPO4,that share 70-82% nucleotide sequence homology, for designing sgRNAs and TALEN binding sites.Molecular analyses and phenotyping of stablePPO-edited transformants. All regeneration events that root on selection media will be advanced for molecular characterization. DNA isolation from the leaves of the selected mutant lines will be performed using DNeasy Plant Mini Kit (Qiagen). PCR screening for targeted mutagenesis along with PCR loss of restriction enzyme site assay (PCR/RE) and T7 endonuclease I assay (T7EI)will be performed to select gene-edited events. Ten independent events will be selected based on these analyses and Sanger sequencing of mutagenized sites. These events will be clonally propagated for three generations to give time for any off-target activity.PPOgene expression in the knock-out lines will be assayed using qRT-PCR and PPO enzyme activity will be quantified by an enzyme-substrate spectrophotometric assay in sliced potato tubers. Phenotyping for PPO activity is an established routine protocol used in the Douches lab. 20μl of tenfold diluted extract will be mixed with 200μl of PPO assay mixture (50mM sodium phosphate buffer pH 6.0, 0.1% SDS and 15mM4-methylcatechol) in a 96-well Microplate. The absorbance reading at A400nmwill be taken in a plate reader every 5 sec for 1min at room temperature. One unit of PPO activity is defined as amount of enzyme required to change the A400nmreading by 0.001/min. For visual phenotyping, potato tubers from each transgenic line and wild type will be randomly selected, cut into equal sections and exposed to air at room temperature.Objective 2:Generate 10 independent knock-out lines ofdelta-12-desaturase(FAD2) inCamelina sativacv. Suneson with all gene editing reagents.Reagent design.CRISPR/Cas9 knock-out constructs targeting theFAD2gene in camelina will be based on constructs used previously. The constructs generated multigenerational, knockouts of all of theFAD2genes in each of the subgenomes in camelina recovering a high oleic acid seed phenotype. The guide RNAs were designed such that they simultaneously targeted all three homoeologousFAD2genes in camelina. Three target sites inFAD2gene were selected to design sgRNAs from the 5' region of the gene with a restriction site upstream of PAM. The same target sites will be used with HypaCas9, Cpf1, base editors and also as TALE binding sites in TALENs constructs. These constructs will be co-expressed withDsRed2,red fluorescence gene along with the nuclease expression. Expression ofDsRed2would allow for easy detection of red fluorescing transgenic T1 seed produced from T0 plants by transformation of camelina using a hand-held green fluorescent flashlight.Validation of reagents via transient expression inArabidopsisprotoplasts. Camelina is closely related toArabidopsisandFAD2gene in both species has highly sequence homology. A well established protocol that has been extensively tested for protoplast isolation and PEG-calcium transfection is established inArabidopsisthat has >90% transfection efficiency. Protoplast transfections will be carried out inArabidopsisprotoplasts using all the genome-editing reagents designed for camelina as described previously. Targeted mutagenesis will be evaluated by PCR screening to identify deletion in mutagenized amplicons along with PCR/RE assay and T7EI assay. Efficiency of all the genome-editing reagents for targeted mutagenesis will be assessed by target specific amplification and sequencing.Phenotyping of edited camelina lines. Analysis of camelina seed fatty acid composition in the positive genome-edited events will be performed as described previously. Total fatty acid content of seeds will be determined in every generation before progressing to the next generation. Trimethylsulphonium hydroxide (TMSH) will be used for transesterification of the fatty acid methyl esters (FAMEs). In brief, single seeds from transgenic camelina plants will be directly crushed in 50 µL of TMSH in glass GC vials before the addition of 400 µL of heptane. Sample vials will be agitated at room temperature for one hour and FAMEs will be analyzed by gas chromatography.Objective 3: Compare specificity of genome-editing reagents using whole genome resequencing and evaluate genomewide off-target frequencies across reagents and crops.Sequence generation.DNA will be isolated from young, dark-treated leaves of each parent, control, and edited potato and camelina line using the Qiagen DNeasy Plant Mini Kit. DNA will be sheared to 500 bp using a Covaris acoustic shearing device and Tru-Seq Nano DNA libraries constructed. Libraries will be sequenced to 25X coverage (150 nt paired end reads) at the MSU Research Technology Support Facility Genomics Core on the Illumina HiSeq 4000 platform. Reads will be assessed using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for quality and then cleaned for quality and adaptors using Cutadapt.Identification of genome variants in edited lines.We will use two bioinformatics complementary approaches to annotate variants in the genome associated with each reagent, transformation, or natural variation. First, we will use theNeedlein thek-stack (NIKS)method outlined inthat relies on a simple comparison ofK-mers between whole genome sequence reads of two individuals to identify variants or mutations. The method does not require a reference genome in that aK-mer frequency histogram is first generated, and sample-specific overlappingK-mers are then identified which are used to seed a local assembly thereby identifying the causal mutation or variant. The NIKS method was used to successfully identify ethylmethane sulfonate (EMS) point mutations in rice andArabis alpina,a species that lacks a reference genome. NIKS was also successful in identifying large-scale irradiation-induced mutations in rice. For this project, we will compare each of the potato or camelina whole genome resequencing datasets with whole genome sequencing reads from the untransformed parental line. Local assemblies of mutations/variants will be aligned to the cognate reference genometo determine the location and impact of the mutation.We will also use a traditional bioinformatics method that involves aligning whole genome resequencing reads to a reference genome sequence and identification of variants. Cleaned reads will be aligned in paired-end mode to the reference genome sequences of potato and camelinausing BWA-MEMand saved in binary alignment format (BAM) using SAMTools version 0.1.19. Output will be sorted, deduplicated, and indexed using Picard tools (http://broadinstitute.github.io/picard/). Reads will be subjected to indel realignment and single nucleotide polymorphisms (SNPs) and indels called using GATK's Unified Genotyper. Variants not present in the parental genome will be attributed to the reagent and/or transformation method.Efficiency and specificity of reagents.We will catalog all variants induced by propagation (natural mutation), somaclonal variation, transformation, and gene editing reagents. We will annotate each variation as (i) SNP, (ii) indel with length of insertion or deletion, (iii) genome location (genic, intergenic, exon, intron, UTR, promoter), (iv) impact on gene function [synonymous, missense, large-impact (frameshift, premature stop, major deletion], and (v) target vs. off-target.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Government regulators of genetically modified organisms, plant geneticists and plant breeders. Changes/Problems:Low transformation and editing efficiency with the original genotypes and constructs selected for potato and camelina required exploring new material and optimizing vectors. We are developingalternate methods of deploying the gene editing reagent in potato (e.g. protoplast regeneration). What opportunities for training and professional development has the project provided?Thilani Jayakody is defending her PhD thesis and has accepted a position as a postdoctoral researcher at Aarhus University, Denmark. She will be developing genome editing methods for legume crops. Thilani Jayakody mentored two undergraduate students, Samantha Sikora and Travis Young in plant transformation, molecular biology and bioinformatics. Samantha Sikora conducted research exploring self-fertility response in 1S1 as contribution the DM1S1/1S1 assembly project which she presented as a poster for Michigan State University's Undergraduate Research Symposium. Samantha Sikora is now continuing work in plant transformations as a research technician at Ball Horticulture. Travis Young is pursuing a summer internship in plant transformations and genome editing with Corteva. At the University of Florida, graduate student Larissa Laforest and undergraduate students Megan Tachev and Trisha Mutisya are getting trained in this project. How have the results been disseminated to communities of interest?Thilani Jayakody published a paper in January 2023titled "Genome Report: Genome sequence of 1S1, a transformable and highly regenerable diploid potato for use as a model for gene editing and genetic engineering." in G3. She also presented this research at PAG30 in San Diego. A manuscript for the WGS off-target study for camelina and potato is preparation. Jayakody et al.'s manuscript "Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering." was awarded the outstanding paper of the year for the American Journal of Potato Research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Genetic transformations in Solanum tuberosum diploid clone DRH195 with Cas9 constructs targeting both alleles of PPO1-3 yielded 27 transgenic events, 7 of which had detectable edits through Sanger sequencing of PCR amplicons and indel decomposition analysis using Synthego's ICE algorithm. Deep amplicon sequencing was conducted to identify allelic contributions of PCR amplicons, supporting the results from the Sanger sequencing. In all cases, except for 1 event, only one allele of on PPO homolog contained an edit. The remaining edit indicated mosaic somatic edits with a mixture of insertion and deletions in one allele of PPO1 and PPO3. Empty vector transformations were also conducted for controls yielding 5 events. Objective 2: Genetic transformations by floral dip method in Camelina sativa cv Suneson were performed by different gene editing reagent constructs. CsFAD2-Cas9 knockout events of nine independent lines created by Jiang et al., 2017 have been molecularly characterized and re-sequenced to detect off-targets. In addition, empty vector transformation controls with DsRed expression cassette and without any gene-editing reagents have been generated. Seven independent transformation control events have been validated for the presence of transgene and re-sequenced for evaluating off-target effects in Obj 3. In addition, in 87 plants using CsFAD2-TALENs construct, 42 plants using CsFAD2-HypaCas9, 25 plants using base editor and 20 plants using Cas12a based gene-editing reagents. DsRed marker expression in the seeds was used to identify the transformed events and further screened for edits using PCR amplification and sanger sequencing. 434 DsRed positive seeds for TALENS, 155 seeds for HypaCas9 were thus far screened for edits. But none of these events had edits in the CsFAD2 region. Tracking of indels by decomposition (TIDE) method was used to analyze the Sanger reads using Cas9-KOs as controls to identify the edits. 50% of the transformed plants have stunted growth and did not make it to seed and some died after germination. We only selected healthy lines that made it to seed to screen for edits. However due to absence of any edits in the target region, we hypothesized that FAD2 KO of all homeologs may have been lethal which is responsible for this lack of growth phenotype. Objective 3: We have completed the genome assembly and annotation of the C. sativa Suneson genome. Using ~42x coverage Oxford Nanopore Technology genomic reads greater than 15 kb and the Flye assembler software, we assembled 647,473,868 bp of the Suneson genome in 551 contigs with an N50 of 12,024,690 bp. Two rounds of error correction with Medaka followed by two rounds of Pilon were performed and then the assembly was filtered to remove contigs less than 50 kb, yielding a 644,482,469 bp assembly in 157 contigs with an N50 length of 12,031,512 bp. To assemble to the 20 camelina chromosomes, Ragtag was used with the DH55 reference assembly resulting in 98.3% of the Suneson assembly anchored to the chromosomes. To validate the assembly, we used KAT to determine the representation of WGS-derived k-mers in the final assembly; the majority of k-mers were present in single copy within the assembly with limited numbers present at two copies and a small set of k-mers present in three copies. To assess representation of genic sequences we ran BUSCO with the embryophyta_odb10 database. A total of 1,606 of the 1,614 (99.5%) BUSCO orthologs were complete in the Sunseson assembly, of which, 1,581 (98.0%) are duplicated as expected due to the hexaploid nature of the camelina genome; a mere 0.2% and 0.3 % were fragmented or missing, respectively. Repetitive sequences in the Suneson genome were identified using a combination of de novo repeat identification and similarity to existing Viridiplantae repetitive sequences. In total, 44.2% of the genome was annotated as repetitive. Retroelements (25%) dominated the annotated repetitive sequences relative to DNA transposons (2.85%). Annotation of protein-coding genes using five mRNA-seq libraries and full-length cDNA sequences from eight different tissues resulted in 145,971 working gene models from 103,435 loci. Of these working models, 133,355 were high confidence models representing 91,877 loci. To assess the quality of the genome annotation, we examined the representation of BUSCO orthologs; within the representative high confidence gene model set, 98.4% of the BUSCO orthologs were present with 92.8% present as duplicated, consistent with the hexaploid nature of the camelina genome. A synthetic assembly for DRH195 was constructed leveraging publically available genomes and sequencing data. The parents of DRH195 include the homozygous diploid potato reference DM and a heterozygous diploid clone RH, each of which have chromosome scale genome assemblies. A previous mapping population generated WGS reads for DRH195 which was aligned to the DM reference genome to identify RH haplotype specific SNP markers. The two RH haplotypes were also aligned to DM to identify SNP markers that discern the two haplotypes. The DRH195 and RH haplotype SNP marker sets were compared to each other to assign the RH haplotype present in DRH195. It was assumed since DM is homozygous that all chromosomes were inherited. A reference sequences was created using a custom python script incorporating RH marker data and reference sequences. All camelina and potato Cas9 and empty vector events were submitted for whole genome sequencing using 2x150bp reads yielding ~60x and ~55x coverage respectively. Variant analysis using WGS was conducted. Preliminary analysis of variants indicate negligible evidence of Cas9 dependent off-targeting in both camelina and potato. The majority of genetic variation was attributed background variation. Mosaic somatic mutations were present in both potato and camelina target sites due to constitutive Cas9 expression. Evidence of transgenerational editing was detected in camelina in Cas9 events into the T5 and T6 generations. A putative translocation was also detected in one camelina Cas9 event, but further investigation into this structural variant is necessary.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Jayakody, T. B., Hamilton, J. P., Jensen, J., Sikora, S., Wood, J. C., Douches, D. S., & Buell, C. R. (2023). Genome Report: Genome sequence of 1S1, a transformable and highly regenerable diploid potato for use as a model for gene editing and genetic engineering. G3: Genes, Genomes, Genetics, 13(4),jkad036.
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Fang, C., Hamilton, J.P., Vaillancourt, B., Wang, Y.-W., Wood, J.C., Deans, N.C., Scroggs, T., Carlton, L., Mailloux, K., Douches, D.S., Nadakuduti, S.S., Jiang, J., and Buell,C.R. Cold stress induces differential gene expression of retained homeologs in Camelina sativa cv Suneson. Under review, Frontiers in Plant Science.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Thilani B. Jayakody, J. Hamilton, J.Wood, J.Jenson, S.Sikora, D. Douches, C. Robin Buell (2022). The genome sequence for a transformable and highly regenerable diploid potato. Potato Association of America Annual Conference. American Journal of Potato Research
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Nadakuduti,S. 2022. Advances in Genome Editing with CRISPR Systems and Transformation Technologies for Plant DNA Manipulation. Frontiers in Plant Science 11:637159. doi: 10.3389/fpls.2020.637159


Progress 09/01/18 to 08/31/23

Outputs
Target Audience:Government regulators of genetically modified organisms, plant geneticists and plant breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?2019-2021: NSF-NRT-IMPACTS traineeship for Thilani Jayakody ($34,000 + tuition and fees for 21 credits) Thilani Jayakody joined the curriculum committee for NSF-NRT-IMPACTS program as graduate student representative Thilani Jayakody was selected to represent IMPACTS program at American Society of Plant Biologists 2021 NSF NRT Traineeship workshop with a presentation on reflections and developments of an interdisciplinary graduate program ($500) Thilani Jayakody awarded National Association of Plant Breeders Borlaug Scholarship ($500) Thilani Jayakody awarded The Jason and Dana Lilly Plant Breeding, Genetics and Biotechology Scholarship to attend Cold Spring Harbor Genome editing conference in 2022 ($1,500) Thilani Jayakody awarded Carter Harrison Endowed Scholarship ($5,000) Thilani Jayakody awarded Future Academic Scholar in Teaching Fellowship ($2,000) Thilani Jayakody awarded 2nd place (tie) for Frank L. Haynes Graduate Student Competition at the 2021 Potato Association of America Annual Conference ($750) At the University of Florida postdoc Dr. Keun Cho, Graduate student Larissa Laforest and undergraduate students Megan Tachev and Trisha Mutisya are being trained in this project. 2022: PhD Student Thilani Jayakody, leads Objectives 1 and 3 for the project. She was awarded a travel grant from NovoCrops ($1000) and the Elmer C. Rossman Endowment Graduate Student Support Award ($2500) to attend the NovoCrop's PhD training on "Biotechnological Advances for Future Crop Production" from August 15, 2022 to August 19, 2022 at the University of Copenhagen, Denmark. Thilani Jayakody mentored two undergraduate students, Jacob Jenson and Samantha Sikora in plant transformation, molecular biology and bioinformatics. Jacob Jenson is continuing education as a PhD student at the University of Minnesota in the Biochemistry, Molecular Biology and Biophysics program, hoping to continue work in synthetic biology. Samantha Sikora conducted research exploring self-fertility response in 1S1 as contribution the DM1S1/1S1 assembly project which she aimed to present as a poster for Michigan State University's Mid-SURE symposium At the University of Florida, graduate student Larissa Laforest and undergraduate students Megan Tachev and Trisha Mutisya are getting trained in this project. 2023: Thilani Jayakody is defending her PhD thesis and has accepted a position as a postdoctoral researcher at Aarhus University, Denmark. She will be developing genome editing methods for legume crops. Thilani Jayakody mentored two undergraduate students, Samantha Sikora and Travis Young in plant transformation, molecular biology and bioinformatics. Samantha Sikora conducted research exploring self-fertility response in 1S1 as contribution the DM1S1/1S1 assembly project which she presented as a poster for Michigan State University's Undergraduate Research Symposium. Samantha Sikora is now continuing work in plant transformations as a research technician at Ball Horticulture. Travis Young is pursuing a summer internship in plant transformations and genome editing with Corteva. How have the results been disseminated to communities of interest?2019-2021: Thilani Jayakody has presented oral talks at the Corteva-PBGB Genome Editing Applications in Plant Breeding Symposium and the Potato Association of America Annual conference. Thilani Jayakody will be submitting a manuscript to the American Journal of Potato Research titled "Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering". Jacob Jenson has presented a poster at the University Undergraduate Research and Arts Forum. Thilani Jayakody and Jacob Jenson were invited for a radio interview for the IMPACTS 88.9 FM show, SciFiles, aired on 6/13/2021. Larissa Laforest presented a poster on this work at the "Breeding and Genomics in Specialty Crops" Annual Plant Science symposium 2021at University of Florida. 2022: Thilani Jayakody published a paper in January 2022 titled "Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering" in the American journal of Potato Research. A figure from this manuscript was selected as the cover of the issue. She also has drafted a manuscript for the DM1S1/1S1 genome assembly work to be submitted as a genome report to G3 titled "The genome sequence for a transformable and highly regenerable diploid potato". Thilani Jayakody was awarded a travel grant from the NSF-NRT IMPACTS program (NSF DGE-1828149, $600) to present a research talk on the work from "Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering" and "The genome sequence for a transformable and highly regenerable diploid potato" at the Potato Association of America meeting in Missoula Montana on July 18 2022 Thilani Jayakody will present the work from "Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering" and "The genome sequence for a transformable and highly regenerable diploid potato" projects during the "Crop improvement in the gene-editing era" symposium at the University of Copenhagen, Denmark on August 18 2022. Thilani Jayakody was selected as the National Potato Council's annual Potato LEAF scholarship recipient for 2021-2022 ($10,000). Thilani was acknowledged for her award with a Q&A article in the industry SpudMan Magazine, and interview on the "Eyes on Potatoes" podcast as well as recognition at the Michigan Winter Potato Conference. Thilani Jayakody was featured in an article in SpudMan Magazine for her Frank L. Haynes Graduate Student Award during the 2021 Potato Association of America Meeting. Thilani Jayakody was selected to represent the Michigan Potato Industry Commission on an economic trade mission to the Netherlands in June 2022 to bolster international ties in sustainable potato breeding efforts. Larissa presented a poster on this work at the "Breeding and Genomics in Specialty Crops" Annual Plant Science symposium 2021at University of Florida. 2023: Thilani Jayakody published a paper in January 2023 titled "Genome Report: Genome sequence of 1S1, a transformable and highly regenerable diploid potato for use as a model for gene editing and genetic engineering." in G3. She also presented this research at PAG30 in San Diego. A manuscript for the WGS off-target study for camelina and potato is preparation. Jayakody et al.'s manuscript "Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering." was awarded the outstanding paper of the year for the American Journal of Potato Research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1.Transformations with 1S1 yielded one Cas9 event with a chimeric edit in PPO1. We recognized Kanamycin antibiotic resistance in 1S1 masked it's transformation capability. We tested transformation efficiency in 1S1 using alternative antibiotics and identified Hygromycin at 10 mg/L elicits a much stronger response.Amplicon sequencing method to screen for edits was developed. We also identified, and obtained dicot codon optimized cytosine base editing constructs containing a Cas9 nickase and UGI. A custom python script was created to identify conserved regions among orthologs and alleles that would create a loss of function when targeted with a cytosine base editor.Genetic transformations in Solanum tuberosum diploid clone DRH195 with Cas9 constructs targeting both alleles of PPO1-3 yielded 27 transgenic events, 7 of which had detectable edits through Sanger sequencing of PCR amplicons and indel decomposition analysis using Synthego's ICE algorithm. Deep amplicon sequencing was conducted to identify allelic contributions of PCR amplicons, supporting the results from the Sanger sequencing. In all cases, except for 1 event, only one allele of on PPO homolog contained an edit. The remaining edit indicated mosaic somatic edits with a mixture of insertion and deletions in one allele of PPO1 and PPO3. Empty vector transformations were also conducted for controls yielding 5 events. We have one peer-reviewed publication from this work and will submit another one in January 2024. Objective 2.Genetic transformations by floral dip method in Camelina sativa cv Suneson were performed by different gene editing reagent constructs. CsFAD2-Cas9 knockout events of nine independent lines created by Jiang et al., 2017 have been molecularly characterized and re-sequenced to detect off-targets. In addition, empty vector transformation controls with DsRed expression cassette and without any gene-editing reagents have been generated. Seven independent transformation control events have been validated for the presence of transgene and re-sequenced for evaluating off-target effects in Obj 3. In addition, in 87 plants using CsFAD2-TALENs construct, 42 plants using CsFAD2-HypaCas9, 25 plants using base editor and 20 plants using Cas12a based gene-editing reagents. DsRed marker expression in the seeds was used to identify the transformed events and further screened for edits using PCR amplification and sanger sequencing. 434 DsRed positive seeds for TALENS, 155 seeds for HypaCas9 were thus far screened for edits. But none of these events had edits in the CsFAD2 region. Tracking of indels by decomposition (TIDE) method was used to analyze the Sanger reads using Cas9-KOs as controls to identify the edits. 50% of the transformed plants have stunted growth and did not make it to seed and some died after germination. We only selected healthy lines that made it to seed to screen for edits. However due to absence of any edits in the target region, we hypothesized that FAD2 KO of all homeologs may have been lethal which is responsible for this lack of growth phenotype. We expect to submita peer-reviewed publication from this objective. Objective 3: We have completed the genome assembly and annotation of the C. sativa Suneson genome. Using ~42x coverage Oxford Nanopore Technology genomic reads greater than 15 kb and the Flye assembler software, we assembled 647,473,868 bp of the Suneson genome in 551 contigs with an N50 of 12,024,690 bp. Two rounds of error correction with Medaka followed by two rounds of Pilon were performed and then the assembly was filtered to remove contigs less than 50 kb, yielding a 644,482,469 bp assembly in 157 contigs with an N50 length of 12,031,512 bp. To assemble to the 20 camelina chromosomes, Ragtag was used with the DH55 reference assembly resulting in 98.3% of the Suneson assembly anchored to the chromosomes. To validate the assembly, we used KAT to determine the representation of WGS-derived k-mers in the final assembly; the majority of k-mers were present in single copy within the assembly with limited numbers present at two copies and a small set of k-mers present in three copies. To assess representation of genic sequences we ran BUSCO with the embryophyta_odb10 database. A total of 1,606 of the 1,614 (99.5%) BUSCO orthologs were complete in the Sunseson assembly, of which, 1,581 (98.0%) are duplicated as expected due to the hexaploid nature of the camelina genome; a mere 0.2% and 0.3 % were fragmented or missing, respectively. Repetitive sequences in the Suneson genome were identified using a combination of de novo repeat identification and similarity to existing Viridiplantae repetitive sequences. In total, 44.2% of the genome was annotated as repetitive. Retroelements (25%) dominated the annotated repetitive sequences relative to DNA transposons (2.85%). Annotation of protein-coding genes using five mRNA-seq libraries and full-length cDNA sequences from eight different tissues resulted in 145,971 working gene models from 103,435 loci. Of these working models, 133,355 were high confidence models representing 91,877 loci. To assess the quality of the genome annotation, we examined the representation of BUSCO orthologs; within the representative high confidence gene model set, 98.4% of the BUSCO orthologs were present with 92.8% present as duplicated, consistent with the hexaploid nature of the camelina genome. A synthetic assembly for DRH195 was constructed leveraging publically available genomes and sequencing data. The parents of DRH195 include the homozygous diploid potato reference DM and a heterozygous diploid clone RH, each of which have chromosome scale genome assemblies. A previous mapping population generated WGS reads for DRH195 which was aligned to the DM reference genome to identify RH haplotype specific SNP markers. The two RH haplotypes were also aligned to DM to identify SNP markers that discern the two haplotypes. The DRH195 and RH haplotype SNP marker sets were compared to each other to assign the RH haplotype present in DRH195. It was assumed since DM is homozygous that all chromosomes were inherited. A reference sequences was created using a custom python script incorporating RH marker data and reference sequences. All camelina and potato Cas9 and empty vector events were submitted for whole genome sequencing using 2x150bp reads yielding ~60x and ~55x coverage respectively. Variant analysis using WGS was conducted. Preliminary analysis of variants indicate negligible evidence of Cas9 dependent off-targeting in both camelina and potato. The majority of genetic variation was attributed background variation. Mosaic somatic mutations were present in both potato and camelina target sites due to constitutive Cas9 expression. Evidence of transgenerational editing was detected in camelina in Cas9 events into the T5 and T6 generations. A putative translocation was also detected in one camelina Cas9 event, but further investigation into this structural variant is necessary. Two peer-revieweed publications resulted from this objective.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Fang, C., Hamilton, J.P., Vaillancourt, B., Wang, Y.-W., Wood, J.C., Deans, N.C., Scroggs, T., Carlton, L., Mailloux, K., Douches, D.S., Nadakuduti, S.S., Jiang, J., and Buell,C.R. Cold stress induces differential gene expression of retained homeologs in Camelina sativa cv Suneson. Sec. Plant Genetics, Epigenetics and Chromosome Biology Volume 14 - 2023 | https://doi.org/10.3389/fpls.2023.1271625
  • Type: Theses/Dissertations Status: Published Year Published: 2023 Citation: Jayakody, Thilani. 2023. EVALUATION AND DEVELOPMENT OF RESOURCES FOR GENOME EDITING AND GENETIC ENGINEERING IN SOLANUM TUBEROSUM. A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Breeding, Genetics and BiotechnologyCrop and Soil Sciences Doctor of Philosophy


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Government regulators of genetically modified organisms, plant geneticists and plant breeders. Changes/Problems:Low transformation and editing efficiency with the original genotypes and constructs selected for potato and camelina required exploring new material and optimizing vectors. What opportunities for training and professional development has the project provided?PhD Student Thilani Jayakody, leads Objectives 1 and 3 for the project. She was awarded a travel grant from NovoCrops ($1000) and the Elmer C. Rossman Endowment Graduate Student Support Award ($2500) to attend the NovoCrop's PhD training on "Biotechnological Advances for Future Crop Production" from August 15, 2022 to August 19, 2022 at the University of Copenhagen, Denmark. Thilani Jayakody mentored two undergraduate students, Jacob Jenson and Samantha Sikora in plant transformation, molecular biology and bioinformatics. Jacob Jenson is continuing education as a PhD student at the University of Minnesota in the Biochemistry, Molecular Biology and Biophysics program, hoping to continue work in synthetic biology. Samantha Sikora conducted research exploring self-fertility response in 1S1 as contribution the DM1S1/1S1 assembly project which she aimed to present as a poster for Michigan State University's Mid-SURE symposium At the University of Florida, graduate student Larissa Laforest and undergraduate students Megan Tachev and Trisha Mutisya are getting trained in this project. How have the results been disseminated to communities of interest?Thilani Jayakody published a paper in January 2022 titled "Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering" in the American journal of Potato Research. A figure from this manuscript was selected as the cover of the issue. She also has drafted a manuscript for the DM1S1/1S1 genome assembly work to be submitted as a genome report to G3 titled "The genome sequence for a transformable and highly regenerable diploid potato". Thilani Jayakody was awarded a travel grant from the NSF-NRT IMPACTS program (NSF DGE-1828149, $600) to present a research talk on the work from "Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering" and "The genome sequence for a transformable and highly regenerable diploid potato" at the Potato Association of America meeting in Missoula Montana on July 18 2022 Thilani Jayakody will present the work from "Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering" and "The genome sequence for a transformable and highly regenerable diploid potato" projects during the "Crop improvement in the gene-editing era" symposium at the University of Copenhagen, Denmark on August 18 2022. Thilani Jayakody was selected as the National Potato Council's annual Potato LEAF scholarship recipient for 2021-2022 ($10,000). Thilani was acknowledged for her award with a Q&A article in the industry SpudMan Magazine, and interview on the "Eyes on Potatoes" podcast as well as recognition at the Michigan Winter Potato Conference. Thilani Jayakody was featured in an article in SpudMan Magazine for her Frank L. Haynes Graduate Student Award during the 2021 Potato Association of America Meeting. Thilani Jayakody was selected to represent the Michigan Potato Industry Commission on an economic trade mission to the Netherlands in June 2022 to bolster international ties in sustainable potato breeding efforts. Larissa presented a poster on this work at the "Breeding and Genomics in Specialty Crops" Annual Plant Science symposium 2021at University of Florida. What do you plan to do during the next reporting period to accomplish the goals? Complete transformations and screening of transgenic potato events in DMRH195 and 1S1 Re-sequence confirmed edited events for potato and camelina Complete development of k-mer based mutation screening Benchmark alignment and k-mer based methods for detection of variants generated through gene editing.

Impacts
What was accomplished under these goals? Objective 1: Transformations in tetraploid potato Atlantic yielded three Cas12a, one Hyper-accurate Cas9, and one Cas9 PCR confirmed transgenic events. None of these events had detectable edits through either a pooled amplicon sequencing or a semi-nested PCR sanger sequencing approach. Expression of reagents in transgenic events were confirmed through RT-PCR, indicating a low editing efficiency in tetraploid potato. Kanamycin antibiotic resistance limited transformations in the originally selected line 1S1. Transformations with a hygromycin resistance (hygR) containing binary vector, pHSE401, yielded 11 PCR confirmed independent events with much tighter selection and fewer escapes. The original binary vectors for Cas9, TALENs, and Hyper-accurate Cas9 were augmented using infusion cloning to include hygR and transformations with Cas9+hygR were conducted in 1S1 yielding 4 putative events. The original binary vectors for Cas9, TALENs and Hyper-accurate Cas9 were also augmented to contain the screenable marker RUBY, and transformations with Cas9 + RUBY were conducted in 1S1 yielding seven independent events In tandem to augmenting original vectors for use in 1S1, a new diploid potato line known to be transformable without resistance to kanamycin and had available genomic resources to expedite experiment design, DMRH195, was selected to begin transformations with original constructs. Reference assemblies for DMRH195's parents, DM(DMv6.1) and RH89-039-16 (RHv3) facilitated PPO ortholog identification, target and putative off-target analysis, as well as design of a semi-nested PCR sanger sequencing approach for screening for edits in DMRH195 events. Transformations in DMRH195 with the original Cas9 vector has yielded four PCR confirmed transgenic events in DMRH195, x2 of which have confirmed edits in a target region. Event 1c has an edit in one allele of PPO1 and event 6b has an edit in one allele of PPO3. Transformations in DMRH195 with the original TALENs vector has currently yielded one putative event. Transformations in DMRH195 with the Cas9+hygR vector has currently yielded one putative event. Objective 2: We sequenced the Celine genome of Camelina using Oxford Nanopore long read sequencing. A de novo draft assembly has been generated using Flye as camelina is a hexaploid so we are currently refining our de novo assembly to generate a more contiguous reference genome for alignment-based detection of off-target edits. CsFAD2-Cas9 knockout events of nine independent lines have been molecularly characterized and re-sequences to detect off-targets. Genetic transformations by floral dip method in Camelina sativa cv Suneson were performed in 87 plants using CsFAD2-TALENs construct, 42 plants using CsFAD2-HypaCas9, 25 plants using base editor and 20 plants using Cas12a based gene-editing reagents. DsRed marker expression in the seeds was used to identify the transformed events and further screened for edits using PCR amplification and sanger sequencing. 434 DsRed positive seeds for TALENS, 155 seeds for HypaCas9 were thus far screened for edits. But none of these events had edits in the CsFAD2 region. Tracking of indels by decomposition (TIDE) method was used to analyze the Sanger reads using Cas9-KOs as controls to identify the edits. 50% of the transformed plants have stunted growth and did not make it to seed and some died after germination. We only selected healthy lines that made it to seed to screen for edits. However due to absence of any edits in the target region, we hypothesized that FAD2 KO may have been lethal which is responsible for this lack of growth phenotype. We pooled leaf samples from 3 such plants and analyzed for edits. Even these lines had no edits proving our hypothesis was incorrect. Empty vector transformation control with DsRed expression cassette and without any gene-editing reagents has been used for transformation of camelina by floral dip method - eight camelina transformation events have been generated and validated for the presence of transgene. As the next step, we will test all the constructs in camelina protoplasts for functionality before we continue with the floral dip transformations. Objective 3:Variant analysis to deduce alternate haplotype of 1S1 was completed. Nine CsFAD2 knockout Cas9 Camelina cv Celine events created by Jiang et al., 2017 were received and re-sequenced using 150bp Illumina reads. An alignment free method of characterizing T-DNA insertion sites was conducted and an alignment based off target analysis is being conducted. The method for alignment free off target analysis is in development. Adaptations to improve accuracy and computational efficiency by removing erroneous k-mers are being incorporated.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Jayakody, T.B., Enciso-Rodr�guez, F.E., Jensen, J. et al. Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering. Am. J. Potato Res. 99, 1324 (2022). https://doi.org/10.1007/s12230-021-09855-y
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2022 Citation: Thilani B. Jayakody, J. Hamilton, J.Wood, J.Jenson, S.Sikora, D. Douches, C. Robin Buell (2022). The genome sequence for a transformable and highly regenerable diploid potato. Potato Association of America Annual Conference.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Nadakuduti,S. 2022. Advances in Genome Editing with CRISPR Systems and Transformation Technologies for Plant DNA Manipulation. Frontiers in Plant Science 11:637159. doi: 10.3389/fpls.2020.637159
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Nadakuduti, S. and F. Enciso-Rodriguez 2022. Advances in Genome Editing with CRISPR Systems and Transformation Technologies for Plant DNA Manipulation. Frontiers in Plant Science 11:637159. doi: 10.3389/fpls.2020.637159


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Government regulators of genetically modified organisms, geneticists and plant breeders. Changes/Problems:Michigan State Universityrestricted hours and the number of personal that could be on campus due to the COVID-19 pandemic.Diploid genotypes selected for this study have significant boundaries to transformation. Although transformable, the efficiency was not sufficient for a project of this scale. Transformation efficiency of UW-W4 was < 1%. We recognized kanamycin antibiotic resistance in diploid potato, 1S1 leading to a transformation efficiency <1%. Due to Covid-19, the number of hours and personnel allowed to be in the lab at a time has been restricted at the University of Florida. We had challenges with Cas12a construct not generating any DsRed seeds after the first round of transformation that involved 21 plants. The promoter driving the DsRed cassette is cruciferin which should be expressed in the seeds and siliques of camelina. We decided to change the promoter driving DsRed to 35S. We also have bar gene included to screen using BASTA herbicide. We established BASTA screening protocol on wild type to use as an alternative screening strategy. What opportunities for training and professional development has the project provided?NSF-NRT-IMPACTS traineeship for Thilani Jayakody ($34,000 + tuition and fees for 21 credits) Thilani Jayakody joined the curriculum committee for NSF-NRT-IMPACTS program as graduate student representative Thilani Jayakody was selected to represent IMPACTS program at American Society of Plant Biologists 2021 NSF NRT Traineeship workshop with a presentation on reflections and developments of an interdisciplinary graduate program ($500) Thilani Jayakody awarded National Association of Plant Breeders Borlaug Scholarship ($500) Thilani Jayakody awarded The Jason and Dana Lilly Plant Breeding, Genetics and Biotechology Scholarship to attend Cold Spring Harbor Genome editing conference in 2022 ($1,500) Thilani Jayakody awarded Carter Harrison Endowed Scholarship ($5,000) Thilani Jayakody awarded Future Academic Scholar in Teaching Fellowship ($2,000) Thilani Jayakody awarded 2ndplace (tie) for Frank L. Haynes Graduate Student Competition at the 2021 Potato Association of America Annual Conference ($750) Thilani Jayakody is mentoring two undergraduate students, Jacob Jenson and Sami Sikora in plant transformation, molecular biology and bioinformatics. Postdoc Dr. Keun Cho, Graduate student Larissa Laforest and undergraduate students Megan Tachev and Trisha Mutisya are being trained in this project. How have the results been disseminated to communities of interest?Thilani Jayakody has presented oral talks at the Corteva-PBGB Genome Editing Applications in Plant Breeding Symposium and the Potato Association of America Annual conference. Thilani Jayakody will be submitting a manuscript to the American Journal of Potato Research titled "Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering". Jacob Jenson has presented a poster at the University Undergraduate Research and Arts Forum. Thilani Jayakody and Jacob Jenson were invited for a radio interview for the IMPACTS 88.9 FM show, SciFiles, aired on 6/13/2021. Larissa Laforest presented a poster on this work at the "Breeding and Genomics in Specialty Crops" Annual Plant Science symposium 2021at University of Florida. What do you plan to do during the next reporting period to accomplish the goals?Continue transformations and screening in Atlantic.Add hygromycin resistance gene to vectors and begin transformations in 1S1 concurrently then begin transformations in 1S1.Develop k-mer based mutation screening method using both simulated and experimentally generated data.Analyze Camelina Cas9 events using K-mer based mutation screening and compare to variant calling based methods.Design sgRNAs and assembly cytosine base editor constructs.Assemble and annotate the Celine WT accession. Benchmark alignment and k-mer-based methods for detection of variants generated through gene editing. Generate gene edited events in camelina using HypaCas9, Cas12a, base editing and TALENs will continue. We will evaluate the DsRed seeds of already transformed plants with TALENS, HypaCas9 plants and perform genetic transformation with Cas12a and base editing constructs as well. We will re-sequence the T3 generation of the gene-edited events using each of the above constructs for the detection of genome-wide off-target effects.

Impacts
What was accomplished under these goals? Objective 1:Transformations with 1S1 yielded one Cas9 event with a chimeric edit in PPO1. We recognized Kanamycin antibiotic resistance in 1S1 masked it's transformation capability. We tested transformation efficiency in 1S1 using alternative antibiotics and identified Hygromycin at 10 mg/L elicits a much stronger response. We are now conducting a transformation test using pHSE401 empty binary vector containing hygromycin resistance gene to uncover true transformation efficiency of 1S1.Transformations using Cas12, TALENs, HypaCas9 and Cas9 began in Atlantic. Amplicon sequencing method to screen for edits was developed. We also identified, and obtained dicot codon optimized cytosine base editing constructs containing a Cas9 nickase and UGI. A custom python script was created to identify conserved regions among orthologs and alleles that would create a loss of function when targeted with a cytosine base editor. Objective 2:In Camelina, CsFAD2 - Cas9 Knock out events were characterized. Ninelines including the WT Camelina sativa cv Celine are being re-sequenced to detect genome-wide off-targets. Specifically, long Oxford Nanopore reads were generated from Celine and Illumina short read sequences are being generated currently for use in error-correction.CsFAD2 - TALENs construct is used to do flora dip with approximately 86 plants of camelina. DsRed marker expressed seeds of some early events are currently being validated for the edits. Most transformed plants are currently growing and will set seed in a month. All of the gene-edited events will subsequently be re-sequenced. CsFAD2 - HypaCas9 construct has been generated and genetic transformation of camelina plants was carried out. The transformed T0 generation 45 plants are currently setting seed. The seed will be screened for DsRed transformation marker and further evaluated for the presence of edits in CsFAD2 gene.Empty vector transformation control construct with DsRed expression cassette and without any gene-editing reagents has been used for transformation of camelina by floral dip method. Eightcamelina transformation events have been generated and validated for the presence of transgene.Cas12a based construct has been generated to knock-out the FAD2 gene and genetic transformation by floral dip method is being carried on. Vectors to design knock-out using cytidine base editors (CBE) and the construction of the final reagent has been accomplished. Base-editing construct using Cas9-PmCDA1-UGI has been generated and currently being sequenced to confirm the final assembly. Genetic transformations will be started using this construct. Fatty Acid Methyl Esters (FAMEs) analyses of camelina seeds and detection of DsRed fluorescence expressed in the seeds has been established for phenotyping of the gene-edited lines. We quantified individual fatty acids in Cas9-knock-out seeds and compared it to the wild type. We confirmed the phenotype as expected by the knock-out of FAD2 gene. Objective 3: Assembly and annotation of DM1S1/1S1 was completed.Concurrently, began work with autotetraploid potato cultivar Atlantic because of previously reported transformation capability, and development of a high quality, reference genome assembly.PPO orthologs have been identified from Atlantic genome assembly and sequences confirmed from Sanger sequencing. We verified conservation in target sequences among most alleles in PPO1-4 in Atlantic. We also verified expression of alleles in tubers and/or leaves using previously generated Atlantic expression data used for genome assembly. Apreliminary off-target analysis was conducted using Cas-Off and custom Atlantic genome and cataloged putative off-targets in assembly. For camelina, we are developing a bioinformatic pipeline to detect off-target events using k-mers from whole genome shotgun sequencing reads thereby alleviating the need for a reference genome and read alignments.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: " Nadakuduti and Enciso-Rodriguez published Advances in Genome Editing with CRISPR Systems and Transformation Technologies for Plant DNA Manipulation. Frontiers in Plant Science 11:637159. doi: 10.3389/fpls.2020.637159
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: USDA  NIFA Project Directors virtual meeting (2021). Oral presentation on Genome Wide Evaluation of Off-Targets from Gene Editing Reagents in Seed vs. Vegetatively Propagated Crop Species
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: " Thilani B. Jayakody, S. Nadakuduti, J.Jenson. F. Enciso-Rodriguez, D. Douches (2021). Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering. American Journal of Potato Research.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: " Thilani B. Jayakody, S. Nadakuduti, J.Jenson. F. Enciso-Rodriguez, D. Douches (2021). Evaluation of diploid potato germplasm for applications of genome editing and genetic engineering. Potato Association of America Annual Conference.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Jacob Jenson, T.B. Jayakody, S. Nadakuduti, D. Douches. (2021). Developing ant-browning tetraploid potatoes using cytosine base editors. Poster. Michigan State University Undergraduate Research and Arts Forum.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: " Thilani B. Jayakody, S. Nadakuduti, C. Robin Buell, D. Douches (2020). Genome Wide Evaluation of Off-targets from Gene Editing Reagents in Solanum tuberosum. Corteva-PBGB Genome Editing Applications in Plant Breeding Symposium. Michigan State University.


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:Government regulators of genetically modified organisms, geneticists and plant breeders. Changes/Problems:In the reporting period, we were highly restricted in our ability to perform laboratory work as of March, 2020 due to the shutdown of research facilities in response to COVID-19. We have been permitted to resume laboratory work, on a limited basis in late June. Due to the requirement for social distancing, we work in shifts to reduce occupancy in the lab. As a consequence, completion of goals for the project have been delayed. What opportunities for training and professional development has the project provided?This project has been a training and professional development for a Ph.D. student. Thilani Jayakody presented her research at the Potato Association of America meeting in July 2019. Jacob Jensen, a Juniorundergraduate was taught basic lab techniques and is now assisting Dr. Nadakuduti and Thilani Jayakody with theirexperiments. How have the results been disseminated to communities of interest?Preliminary results were disseminated at the Potato Association of American meeting in July 2020. Thilani Jayakody gave an oral presentation and Jacob Jensen presented a poster. What do you plan to do during the next reporting period to accomplish the goals?We will continue to complete the objectives to obtain the sets of potato and camelina editing events needed for objective 3. We also have to complete the assembly of the DM1S1 sequence as part of objective 3.

Impacts
What was accomplished under these goals? ?Objective 1 Selection of germplasm: Two diploid self-compatible and homozygous lines US-W4 and 1S1 were selected based on successful regeneration capability in tissue culture. Gene-editing reagent construction: The target gene in potato for gene-editing is polyphenol oxidase (PPO). We have finished the generation of CRISPR/Cas9, Hypa/Cas9, Cas12a, TALENs based gene-editing reagent constructs to knock-out conserved region of all four tuber expressed PPOs, the details of which are given below: We harvested the tubers of W4 and 1S1 diploid lines. In order to quantitatively determine the level of PPO expression within the tuber, the tissue was separated into tuber peel, tuber core and whole tuber. RNA was isolated from the whole tuber tissue of 1S1 and W4 and cDNA was synthesized. Complete ORFs of tuber expressed PPOs (StPPO1 - StPPO4) were cloned and sequenced. Multiple bacterial clones (12 clones for each gene) were sequenced in order to capture the allelic variation within the species. Conserved regions between all PPOs and their alleles in the two potato lines were chosen as targets for CRISPR systems and TALENs. CRISPR- Cas9, HypaCas9 constructs with dual sgRNAs were generated and confirmed by sequencing. An empty vector/ negative control that contains the Cas9 but not sgRNAs was also made. All these reagent plasmids have been transformed into Agrobacterium GV3101 ready for Agrobacterium-mediated transformation. The CRISPR/Cas9 constructs have also been validated for on-target functionality in protoplast system. By using the integration of double stranded oligonucleotide method established in the lab, we were able to successfully detect the on-target activity of single guide RNA2 target genes PPO2 and PPO1 genes in both W4 and 1S1 genotypes. Therefore, single guide RNA2 target has also been used as target for Cas12a constructs as it contained the TTTV PAM required for Cas12a. This reagent plasmid has been transformed into Agrobacterium GV3101. We have designed and generated the TALENS construct based on the conserved regions. Protein based DNA recognition mechanism in TALENs requires that the two TALEN binding sites are 12-25 bp apart which has limited us to design a single (TBS) that could target all PPO genes, however the other TBS would only target PPO2, the most highly expressed PPO in the potato tuber. This construct has been transformed into Agrobacterium GV3101. Phenotyping by using PPO enzyme activity by an enzyme-substrate spectrophotometric assay has been established in both W4 and 1S1 tuber tissues which have shown PPO activity in this assay. The absorbance reading at A400nm in the assay was highest for peel tissue in both lines suggesting that PPO activity is higher in peel than core. Stable transformation of gene edited reagents into diploid potato: All of the generated gene-editing reagents constructs are being used in several rounds of Agrobacterium-mediated transformations of W4 and 1S1. Currently, we are screening multiple events generated from each of these reagents based on the kanamycin resistance marker. Since we aimed at knocking-out all potato tuber expressed PPOs, we were limited by conserved regions as well as protospacer adjacent motif (PAM) presence and no-off targets in those regions. We are currently working on developing a high throughput method of screening for mutations in the target site. The strategy is to multiplex genomic DNA from transformation events for Amplicon-Sequencing. We have also acquired the vectors to design knock-out using cytidine base editors and the construction of the final reagent is in progress. Objective 2 Selection of germplasm: Camelina sativa cv. Suneson has been selected for this project since it has previously shown success in transformation and gene editing. Gene-editing reagent construction: FAD2 is the target gene for knock-out using various reagents in Camelina. Jiang et al., (Plant Biotechnology Journal (2017) 15, pp. 648-657) have generated FAD2 knock-out lines in Camelina. In their study, guide RNAs were designed that simultaneously targeted all three homoeologous FAD2 genes in the hexaploid Camelina. They obtained a significantly enhanced oil composition in T3 and T4 generation Camelina FAD2 knock-out seeds, since FAD2 is involved in fatty acid biosynthesis catalyzing the conversion of mono-unsaturated to polyunsaturated fatty acids. A combination of germ-line mutations and somatic cell mutations in FAD2 genes in each of the three Camelina sub genomes has been achieved. Therefore, we decided to use the already generated Camelina gene-edited lines using CRISPR/Cas9 in our study to detect off-targets. After getting approval for inter-state movement of these seeds by USDA-BRS, we aquired the seeds of 13 different gene-edited FAD2 knock-out lines ranging from T3 - T5 generations based on the best vigor and seed oil phenotype. We are currently sequencing the target region to confirm the edits. These seeds will be used as positive controls for establishing DsRed phenotyping and for Fatty acid analyses. In order to have DsRed fluorescence as a selection marker in the T-DNA backbone vector for phenotyping, we cloned the DsRed expression cassette into the binary vector backbone ready to be used during assembly of other gene-editing reagents to target FAD2 in camelina. To generate the Hypa-Cas9 construct, the same guide RNAs will be used as in the above study. We have generated Hypa-Cas9 vector for generating the potato PPO-Hypa-Cas9 construct, which will also be used for camelina. Golden gate assembly to generate camelina FAD2-HypaCas9 construct is in progress. Based on the sequence analyses of FAD2, coincidentally, out of the three spacers in guide RNAs used in Cas9 construct, two of them also also have TTTV PAM for Cas12a. Therefore, similar regions will also be used for Cas12a construct. However, TALENs construct still need to be designed. We have acquired the vectors to design knock-out using cytidine base editors (CBE) and the construction of the final reagent is in progress. Currently restriction enzyme digests are designed to confirm the identity of the vectors after which the CBE-knock-out construct will be assembled by golden gate/ gateway cloning methods. Empty vector control construct with DsRed expression cassette has been used for transformation of camelina by floral dip method and analyses of the transgenic seeds based on DsRed marker is in progress. Fatty Acid Methyl Esters (FAMEs) analyses of camelina seeds and detection of DsRed fluorescence expressed in the seeds are being established for phenotyping of the gene-edited lines. Objective 3 To facilitate in the screening of off-target effects, we generated a high-quality reference assembly for a doubled monoploid of 1S1 (DM1S1) using oxford nanopore sequencing. High molecular weight gDNA was isolated and purified from DM1S1and sequenced on MinION R9 flowcells. 44 Gb of reads that were greater than 10kb were used for the genome assembly.Reads were assembled using Flye/2.3.2. To polish assembly, we generated 32 Gb of QC filtered 150 bp illumina PE reads. Assembly was polished using Racon/1.3.2, Medaka0.12.1 and Pilon/1.23. The assembly was scaffolded into 12 potato chromosomes using RaGOO/v1. We also generated 71 Gb Illumina 150 bp PE reads of 1S1 to be aligned to DM1S1 assembly in order to resolve the haplotype.

Publications


    Progress 09/01/18 to 08/31/19

    Outputs
    Target Audience:Government regulators of genetically modified organisms, geneticists and plant breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has been a training and professional development for a M.S. student. Thilani Jayakody presented her research at the Potato Association of America meeting in July 2019. Jacob Jensen, a freshman undergraduate was taught basic lab techniques and is now assisting Dr. Nadakuduti with her experiments. How have the results been disseminated to communities of interest?Preliminary results were disseminated at the Potato Association of American meeting in July 2019. What do you plan to do during the next reporting period to accomplish the goals?Our goal is to develop the potato and camelina events that will be anaylized in Objective 3.

    Impacts
    What was accomplished under these goals? The aim of this project is to use a diploid potato line that is vegetatively propagated and camelina, a seed propagated species as two different crop systems to compare the level of genome-wide off-targets caused by current genome-editing technologies. We will quantify and compare the genome-wide off-targets caused by CRISPR/Cas9, HypaCas9, CRISPR-Cpf1, base editors, and TALENs in both crop species. This will be compared with mutation rates in a set of control lines that represent mutation associated with tissue culture and regeneration, single seed descent, and transformation. Objective 1: Generate 10 independent PPO knock-out lines in the diploid potato line with five genome-editing reagents Selection of germplasm: Two diploid self-compatible and homozygous lines US-W4 and 1S1 were tested for regeneration capability in tissue culture and showed successful regeneration. Gene-editing reagent construction: The target gene in potato for gene-editing is polyphenol oxidase (PPO). PPOs belong to multigene family that are highly conserved, primarily clustered in tandem or in close proximity to each other. In order to generate gene-editing reagent constructs to knock-out conserved region of tuber expressed PPOs: We harvested the tubers of W4 and 1S1 diploid lines. In order to quantitatively determine the level of PPO expression within the tuber, the tissue was separated into tuber peel, tuber core and whole tuber. RNA was isolated from the whole tuber tissue of 1S1 and W4 and cDNA was synthesized in order to clone the tuber expressed PPOs Complete ORFs of tuber expressed PPOs (StPPO1 - StPPO4) were cloned and sequenced. Multiple bacterial clones were sequenced in order to capture the allelic variation within the species Conserved regions between all PPOs and their alleles in the two potato lines were chosen as targets for CRISPR systems and TALENs. Two CRISPR- Cas9 constructs with a single spacer sequence each and one with dual sgRNAs are constructed and confirmed by sequencing. Empty vector/ negative control that contains the Cas9 but not sgRNAs was also made. All these reagent plasmids have been put into Agrobacterium GV3101 ready for Agrobacterium mediated transformation. The same constructs have also been cloned into protoplast vectors for validation of reagents via transient protoplast expression. The same conserved regions in PPO have also been used as target sequences for Cas12a constructs. Two CRISPR-Cas12a constructs are being generated for Agrobacterium mediated transformation. Validation of reagents via transient protoplast expression: US-W4 and 1S1 diploid lines have been propagated in tissue culture for protoplast isolation and transfection with reagent plasmid vectors (Cas9 and Cas12a) generated. Protoplast isolation and transfection protocol has been established in the lab. Once validated stable transformation using Agrobacterium will be performed. We are currently working on designing HypaCas9 variant. The strategy is to order a gBLOCK that consists of all the variations pertaining to HypaCas9 and assemble into one of the vectors via Gibson assembly. Assembling the final Cas12a constructs targeting PPOs in potato TALENs targeting PPOs in potato Tissue culture propagation to increase W4 and 1S1 for protoplast validation of constructs Phenotyping of PPO-edited lines Phenotyping by using PPO enzyme activity by an enzyme-substrate spectrophotometric assay has been established. Both W4 and 1S1 tuber tissues have shown PPO activity in this test. The absorbance reading at A400nm in the assay was highest for peel tissue in both lines suggesting that PPO activity is higher in peel than core. Objective 2: Generate 10 independent knock-out lines of delta-12-desaturase (FAD2) in Camelina sativa cv. Suneson with all gene editing reagents. Suneson cultivar seeds have been obtained and plants were grown. Single seed descent seeds were isolated for experiments to avoid any mixing up since the study involves whole genome sequencing. We are currently working on designing reagents for targeted knock-out of FAD2 in Camelina using CRISPR/Cas9, Cas12a, HypaCas9, TALENs Objective 3 will not be initiated until years 2 and 3 of the project.

    Publications