Multiplex CRISPR/Cas9 gene-editing platform in oil palm targeting mutations in EgFAD2 and EgPAT genes
Tóm tắt
CRISPR/Cas9 is the most powerful and versatile genome-editing tool that permits multiplexed-targeted gene modifications for the genetic enhancement of oil palm. Multiplex genome-editing has recently been developed for modifying multiple loci in a gene or multiple genes in a genome with high precision. This study focuses on the development of high-oleic oil palm, the primary target trait for healthy low-saturated oil. To achieve this, the fatty acid desaturase 2 (FAD2) and palmitoyl-acyl carrier protein thioesterase (PAT) genes, both of which are associated with fatty acid metabolism biosynthesis pathways in oil palm, need to be knocked out. The knockout of FAD2 and PAT leads to an accumulation of oleic acid content in oil palms. A total of four single-guide RNAs (sgRNAs) were designed in silico based on the genomic sequences of EgFAD2 and EgPAT. Using robust plant CRISPR/Cas9 vector technology, multiple sgRNA expression cassettes were efficiently constructed into a single-binary CRISPR/Cas9 vector to edit the EgFAD2 and EgPAT genes. Each of the constructed transformation vectors was then delivered into oil palm embryogenic calli using the biolistic, Agrobacterium-mediated, and PEG-mediated protoplast transformation methods. Sequence analysis of PCR products from 15 samples confirmed that mutations were introduced at four target sites of the oil palm EgFAD2 and EgPAT genes. Single- and double-knockout mutants of both genes were generated, with large and small deletions within the targeted regions. Mutations found at EgFAD2 and EgPAT target sites indicate that the Cas9/sgRNA genome-editing system effectively knocked out both genes in oil palm. This technology is the first in oil palm to use CRISPR/Cas9 genome-editing to target high-oleic-associated genes. These findings showed that multiplex genome-editing in oil palm could be achieved using multiple sgRNAs. Targeted mutations detected establish that the CRISPR/Cas9 technology offers a great potential for oil palm.
Tài liệu tham khảo
Parveez GKA, Tarmizi AHA, Sundram S, Loh SK, Ong-Abdullah M, Palam KDP, Salleh KM, Ishak SM, Idris Z (2021) Oil palm economic performance in Malaysia and R&D progress in 2020. J Oil Palm Res 33(2):181–214. https://doi.org/10.21894/jopr.2021.0026
Sambanthamurthi R, Sundram K, Tan YA (2000) Chemistry and biochemistry of palm oil. Prog Lip Res 39:507–558
Sundram K, Sambanthamurthi R, Tan YA (2003) Palm fruit chemistry and nutrition. Asia Pac J Clin Nutr 12:355–362
Pedersen JI, Muller H, Seljeflot I, Kirkhus B (2005) Palm oil versus hydrogenated soybean oil: effects on serum lipids and plasma haemostatic variables. Asia Pac J Clin Nutr 14(4):348–357
Lopez-Huertas E (2010) Health effects of oleic acid and long chain omega-3 fatty acids (EPA and DHA) enriched milks. A review of intervention studies. Pharmacol Res 61:200–207
Parveez GKA, Rasid OA, Masani MYA, Sambanthamurthi R (2015) Biotechnology of oil palm: strategies towards manipulation of lipid content and composition. Plant Cell Rep 34:533–543
Masani MYA, Izawati AMD, Rasid OA, Parveez GKA (2018) Biotechnology of oil palm: current status of oil palm genetic transformation. Biocatal Agric Biotechnol 15:335–347. https://doi.org/10.1016/j.pbcab.2018.07.008
Zulkifli Y, Norziha A, Naqiuddin MH, Fadila AM, Nor Azwani AB, Suzana M, Samsul KR, Ong-Abdullah M, Singh R, Parveez GKA, Kushairi A (2017) Designing the oil palm of the future. J Oil Palm Res 29:440–455. https://doi.org/10.21894/jopr.2017.00015
Yunus AMM, Kadir APG (2008) Development of transformation vectors for the production of potentially high oleate transgenic oil palm. Electron J Biotechnol 11(3):23–31. https://doi.org/10.4067/S0717-34582008000300003
Rasid OA, Masura SS, Hanin NA, Masli DIA, Bohari B, Masani MYA, Parveez GKA (2020) Oil palm transgenic research: challenges, update, and future outlook. In: Ithnin M, Kushairi A (eds) The Oil Palm Genome. Compendium of Plant Genomes. Switzerland, Springer Nature, pp 69–76. https://doi.org/10.1007/978-3-030-22549-0_6
Rosli R, Amiruddin N, Ab Halim MA, Chan PK, Chan KL, Azizi N, Morris EP, Leslie ET, Abdullah MO, Sambanthamurthi R, Singh R, Murphy DJ (2018) Comparative genomic and transcriptomic analysis of selected fatty acid biosynthesis genes and CNL disease resistance genes in oil palm. PlosOne. https://doi.org/10.1371/journal.pone.0194792
Singh R, Ong-Abdullah M, Low ETL, Manaf MAA, Rosli R, Nookiah R, Ooi LCL, Ooi SE, Chan KL, Halim MA, Azizi N (2013) Oil palm genome sequence reveals divergence of interfertile species in old and new worlds. Nature 500(7462):335–339
Yeap WC, Norkhairunnisa CMK, Norfadzilah J, Muad MR, Appleton DR, Harikrishna K (2021) An efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 mutagenesis system for oil palm (Elaeis guineensis). Front Plant Sci 12:773656. https://doi.org/10.3389/fpls.2021.773656
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247–271. https://doi.org/10.1146/annurev-genet-110410-132435
Jarvis BA, Romsdahl TB, McGinn MG, Nazarenus TJ, Cahoon EB, Chapman KD, Sedbrook JC (2021) CRISPR/Cas9-induced fad2 and rod1 mutations stacked with fae1 confer high oleic acid seed oil in Pennycress (Thlaspi arvense L.). Front Plant Sci. https://doi.org/10.3389/fpls.2021.652319
Bahariah B, Masani MYA, Rasid OA, Parveez GKA (2021) Multiplex CRISPR/Cas9-mediated genome editing of the FAD2 gene in rice: a model genome editing system for oil palm. J Genet Eng Biotechnol 19:86–86. https://doi.org/10.1186/s43141-021-00185-4
Tian Y, Chen K, Li X, Zheng Y, Chen F (2020) Design of high-oleic tobacco (Nicotiana tabacum L.) seed oil by CRISPR-Cas9-mediated knockout of NtFAD2–2. BMC Plant Biol 20:233
Yuan M, Zhu J, Gong L, He L, Lee C, Han S, Chen C, He G (2019) Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing. BMC Biotechnol 19:24. https://doi.org/10.1186/s12896-019-0516-8
Wu N, Lu Q, Wang P, Zhang Q, Zhang J, Qu J, Wang N (2020) Construction and analysis of GmFAD2-1A and GmFAD2-2A soybean fatty acid desaturase mutants based on CRISPR/Cas9 technology. Int J Mol Sci 21(3):1104. https://doi.org/10.3390/ijms21031104
Do PT, Nguyen CX, Bui HT, Tran LTN, Stacey G, Gillman JD, Zhang ZJ, Stacey MG (2019) Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homoeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and α-linolenic acid phenotype in soybean. BMC Plant Biol 19:311. https://doi.org/10.1186/s12870-019-1906-8
Ayako O, Takumi O, Chie K, Kanako K, Mizue I, Jun I, Nobuya K (2018) CRISPR/Cas9-mediated genome editing of the fatty acid desaturase 2 gene in Brassica napus. Plant Physiol Biochem 131:63–69
Jiang WZ, Henry LM, Lynagh PG, Comai L, Cahoon EB, Weeks DP (2017) Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing. Plant Biotechnol J 15:648–657
Morineau C, Bellec Y, Tellier F, Gissot L, Kelemen Z, Nogue F, Faure JD (2017) Selective gene dosage by CRISPR-Cas9 genome editing in hexaploid Camelina sativa. Plant Biotechnol J 15:729–739
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823
Zuker M (2003) Mfold web server for nucleic aclid folding and hybridization prediction. Nucleic Acids Res 31(13):3406–3434
Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CR, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345
Masani MYA, Noll GA, Parveez GKA, Sambanthamurthi R, Prüfer D (2014) Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. PLoS ONE 9(5):e96831
Masli DIA, Kadir APG, Yunus AMM (2009) Transformation of oil palm using Agrobacterium tumefaciens. J Oil Palm Res 21:643–652
Bahariah B, Masani MYA, Rasid OA, Parveez GKA (2017) Construction of a vector containing hygromycin (HPT) gene driven by double 35S (2XCAMV35S) promoter for oil palm transformation. J Oil Palm Res 29(2):180–188. https://doi.org/10.21894/jopr.2017.2902.03
Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl Acid Res 19:1349. https://doi.org/10.1093/nar/19.6.1349
Hsiau T, Maures T, Waite K, Yang J, Kelso R, Holden K, Stoner R (2018) Inference of CRISPR Edits from Sanger trace data. Biorxiv. 251082.https://doi.org/10.1101/251082
Zuker M, Stiegler P (1981) Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res 9:133–148
Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284
Fizree PMAA, Shaharuddin NA, Ling HC, Masura SS, Manaf MAA, Parveez GKA, Masani MYA (2019) Evaluation of transient DsRED gene expression in oil palm embryogenic calli. Sci Hortic 257:108679. https://doi.org/10.1016/j.scienta.2019.108679
Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507(7490):62–67
Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods 10(11):1116–1121
Ma M, Ye AY, Zheng W, Kong L (2013) A guide RNA sequence design platform for the CRISPR/Cas9 system for model organism genomes. BioMed Res Int 2013:270805
Liang G, Zhang H, Lou D, Yu D (2016) Selection of highly efficient sgRNAs for CRISPR/Cas9 based plant genome editing. Sci Rep 6:21451
Uniyal AP, Mansotra K, Yadav SK, Kumar V (2019) An overview of designing and selection of sgRNAs for precise genome editing by the CRISPR-Cas9 system in plants. 3 Biotech 9(6):223. https://doi.org/10.1007/s13205-019-1760-2
Jamaludin N, Bahariah B, Shaharuddin NA, Ho CL, Rasid OA, Parveez GKA, Masani MYA (2022) Designing gRNAs targeting oil palm phytoene desaturase (EgPDS) gene and construction of vectors for oil palm CRISPR/Cas9 study. J Oil Palm Res. https://doi.org/10.21894/jopr.2022.0032
Thuzar M, Vanavichit A, Tragoonrung S, Jantasuriyarat C (2011) Efficient and rapid plant regeneration of oil palm zygotic embryos cv. “Tenera” through somatic embryogenesis. Acta Physiol Plant 33:123–128. https://doi.org/10.1007/s11738-010-0526-6
Wan NurSyuhada WS, Rasid OA, Parveez GKA (2016) Evaluation on the effects of culture medium on regeneration of oil palm plantlets from immature embryos (IE). J Oil Palm Res 28:234–239. https://doi.org/10.21894/jopr.2016.2802.12
Weckx S, Inzé D, Maene L (2019) Tissue culture of oil palm: finding the balance between mass propagation and somaclonal variation. Front Plant Sci 10:722