First Successful Targeted Mutagenesis Using CRISPR/Cas9 in Stably Transformed Grain Amaranth Tissue

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results First Successful Targeted Mutagenesis Using CRISPR/Cas9 in Stably Transformed Grain Amaranth Tissue View ORCID Profile Susanne K Vollmer , View ORCID Profile Markus G Stetter , View ORCID Profile Götz Hensel doi: https://doi.org/10.1101/2025.02.22.639339 Susanne K Vollmer 1 Heinrich Heine University Du□sseldorf, Faculty of Mathematics and Natural Sciences, Centre for Plant Genome Engineering , Du□sseldorf, 40225, Germany 2 Institute for Plant Sciences, University of Cologne , Cologne, 50674, Germany 3 Cluster of Excellence in Plant Sciences (CEPLAS), Heinrich Heine University Du□sseldorf , 40225, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Susanne K Vollmer Markus G Stetter 2 Institute for Plant Sciences, University of Cologne , Cologne, 50674, Germany 3 Cluster of Excellence in Plant Sciences (CEPLAS), Heinrich Heine University Du□sseldorf , 40225, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Markus G Stetter Götz Hensel 1 Heinrich Heine University Du□sseldorf, Faculty of Mathematics and Natural Sciences, Centre for Plant Genome Engineering , Du□sseldorf, 40225, Germany 3 Cluster of Excellence in Plant Sciences (CEPLAS), Heinrich Heine University Du□sseldorf , 40225, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Götz Hensel For correspondence: henselg{at}hhu.de Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary Genome editing using CRISPR/Cas is a key technology for speeding up breeding for climate-resilient, high-yielding crops ( Scheben et al ., 2017 ). However, efficient targeted mutagenesis requires implementing stable transformation methods and establishing a CRISPR/Cas setup suitable for the species of interest ( Shan et al ., 2020 ). The availability of such methods is a significant bottleneck to advancing many promising, albeit under-researched, crops. Testing and establishing vectors for efficient application of CRISPR/Cas in non-model crops could boost research and breeding of new valuable crops ( Ye and Fan, 2021 ). We edited key pathway genes in the betalain biosynthesis pathway of grain amaranth, i.e., A. hypochondriacus L ., to prove how targeted mutagenesis can be implemented in an orphan crop using the CasCADE modular cloning system ( Hoffie, 2022 ). Grain amaranth is a resilient C 4 dicot orphan crop with excellent nutritional composition. These properties make amaranth a well-suited candidate to be bred as a climate-resilient crop ( Joshi et al ., 2018 ). However, no efficient and reproducible protocol for successful application of CRISPR/Cas9 or stable transformation and regeneration, has been demonstrated in A. hypochondriacus ( Castellanos-Arévalo et al ., 2020 ). Main part Genome editing using CRISPR/Cas is a key technology for speeding up breeding for climate-resilient, high-yielding crops ( Scheben et al ., 2017 ). However, efficient targeted mutagenesis requires implementing stable transformation methods and establishing a CRISPR/Cas setup suitable for the species of interest ( Shan et al ., 2020 ). The availability of such methods is a significant bottleneck to advancing many promising, albeit under-researched, crops. Testing and establishing vectors for efficient application of CRISPR/Cas in non-model crops could boost research and breeding of new valuable crops ( Ye and Fan, 2021 ). We edited key pathway genes in the betalain biosynthesis pathway of grain amaranth, i.e., A. hypochondriacus L ., to prove how targeted mutagenesis can be implemented in an orphan crop using the CasCADE modular cloning system ( Hoffie, 2022 ). Grain amaranth is a resilient C 4 dicot orphan crop with excellent nutritional composition. These properties make amaranth a well-suited candidate to be bred as a climate-resilient crop ( Joshi et al ., 2018 ). However, no efficient and reproducible protocol for successful application of CRISPR/Cas9 or stable transformation and regeneration, has been demonstrated in A. hypochondriacus ( Castellanos-Arévalo et al ., 2020 ). Amaranth produces red and yellow betalains, specialized metabolites in Caryophyllales species ( Timoneda et al ., 2019 ). Betalains have been employed as reporters in molecular biology using the RUBY cassette, which consists of the three enzymes required to produce red betalains from tyrosine ( He et al ., 2020 ). Conversely, the pathway is well suited to evaluate the knock-out efficiency through targeted mutagenesis in A. hypochondriacus , where the pigments naturally occur. Key enzyme genes in the betalain pathway are AhCYP76AD2 and AhCYP76AD5 , which catalyze the initial steps of betalain production. Natural knock-outs of AhCYP76AD2 have been shown to lack red color ( Winkler et al ., 2024 ), suggesting its suitability as a visual reporter. The foundations of successful CRISPR/Cas-mediated targeted mutagenesis are efficient guide RNAs and suitable components to express the Cas9 enzyme and the gRNAs ( Shan et al ., 2020 ). The Cas9 sequence should be adapted to the codon usage of the target species. The promoter to drive the expression of Cas9 and the guides need to be vigorously active in the target species. Finally, the selection cassette has to function efficiently in the target species. To address these requirements in amaranth, we constructed a binary vector containing Cas9 and a four-guide cassette, using the CasCADE modular cloning system ( Figure 1a , Supplemental Figure 1, Supplemental Table 1, Appendix 1; Hoffie, 2022 ). Two guides were designed to target the first exon of AhCYP76AD2 (gRNA 1 and gRNA 2) and one to target the first exon of AhCYP76AD5 (gRNA 4, Figure 1a ). Amaranth plants successfully mutated in AhCYP76AD2 should be deficient in betalains, facilitating a later detection of successfully edited regenerates. An additional gRNA not targeting the amaranth genome, but the CYP76AD2 gene in the RUBY reporter cassette ( He et al ., 2020 ; gRNA 3) was included as control. Download figure Open in new tab Figure 1: Overview of CRISPR/Cas9-mediated targeted mutagenesis in grain amaranth. a) Vector with four gRNA arrays, two targeting the first exon of AhCYP76AD2 (gRNA 1 and gRNA 2), one the first exon of AhCYP76AD5 (gRNA 4) and one the RUBY reporter (gRNA 3). Schematic structure of the target genes with the respective target sites of the guides in amaranth genes. b) Total number of mutations for all three guides. c) Length and frequency of the observed indels found in 74 calli. The Cas9 gene was codon-optimized for the dicot Arabidopsis thaliana and driven by the parsley Ubi4-2 promoter ( Fauser et al ., 2014 ; Hoffie, 2022 ). Each gRNA was expressed separately by the AtU6-26 promoter ( Hoffie, 2022 ). For the transgenic tissue selection, the T-DNA contains an intronized hpt gene driven by a CaMV doubled-enhanced 35S promoter to confer hygromycin resistance ( Hoffie, 2022 ). We cloned the CRISPR/Cas9 cassette into a binary vector via Sfil to enable Rhizobium -mediated transformation ( Hoffie, 2022 ). Next, we established callus transformation, which enables the highly efficient production of stable transgenic tissue in multiple-grain amaranth species. The results were obtained from two independent transformation replicates, batch 1 and 2. A random subset of 10 calli from batch 1 and all from batch 2 treated with R. radiobacter carrying the CRISPR/Cas9 vector were analyzed for edits. Briefly, 5-week-old calli of A. hypochondriacus were transformed with the CRISPR/Cas9 vector using R. radiobacter strain GV3101. For co-culture, calli were incubated on filter paper for three days before being transferred to a callus induction medium with selection. All calli treated with Rhizobium carrying the CRISPR/Cas9 vector (42/42 for batch 1 and 65/65 for batch 2) grew new resistant calli on the selection medium. In contrast, only two (1/21 for batch 1 and 1/32 for batch 2) of the non-treated calli survived the selection and propagated new callus, indicating the high success of transformation. After multiple rounds of selection, callus material was sampled for genotyping and analysis ( Supplemental Figure 2 ). Genomic DNA (gDNA) was extracted from 74 calli of the two independent batches. We sampled individual calli; however, callus tissue can contain cells from independent transformation events with different edits. Therefore, each gDNA included a pool of wild-type and edited alleles. To increase the sensitivity for detecting edited alleles using Sanger sequencing, we employed restriction enzyme digestion-suppressed PCR (RE-PCR) for two guides (gRNA 1 and gRNA 4) in the amaranth genome. Through the deconvolution of the obtained chromatograms, we found edits in 50% (gRNA 4) and 48% (gRNA 1) of the analyzed calli ( Figure 1b ). In contrast, for gRNA 2, a lack of a restriction enzyme recognition site at the Cas9-mediated cut site precluded RE-PCR, edits could only be detected in 12% of the samples. Among all samples with data available for the three sites, 26.8% were edited for both target sites, and 7.1% for all three guide positions. To ensure complete knockouts for downstream analysis of the target gene, targeted mutagenesis should achieve a diverse set of mutations including larger indels. According to the deconvolution data, most edits resulted in a one-base pair deletion or insertion ( Figure 1c ). However, for each guide, edits with larger deletions were also found (max 35 bp for gRNA 4, 17 bp for gRNA 2, 38 bp for gRNA 1; Figure S3 ). To confirm the results from the deconvolution, we cloned the target region of gRNA 4 and gRNAs 1 and 2 from a subset of samples into pGEM-Teasy (Promega) and sequenced multiple colonies. While the frequency of mutations differed, the types of mutations mostly agreed across both methods (despite the restricted sample size of eight sequenced clones from the subcloning) among samples ( Supplemental Table 2 ). Our results show the first successful targeted mutagenesis in grain amaranth using CRISPR/Cas9. Using the CasCADE modular cloning system and our established callus transformation, we edited approximately 50% of all analyzed calli for both target genes. This paves the way for genome editing in grain amaranth for research and breeding of this orphan crop. Moreover, it may also guide the design of CRISPR systems for other species of the Caryophyllales , where reports of successful edits are still scarce. Conflict of interest The authors declare that they have no competing interests. Author Contributions M.G.S. and G.H. supervised the project. S.K.V. performed the experiments and analyzed the data. S.K.V., M.G.S. and G.H. discussed and interpreted the data. S.K.V. wrote the manuscript and prepared the figures. All authors edited and approved the manuscript. Supporting Information Supplemental Figure 1 – Plasmid map of vector used for the targeted mutagenesis (CPGE_VEC00447) Supplemental Figure 2 – Genotyping of calli for the presence of the Cas9 gene Supplemental Figure 3 -Gel electrophoresis of 35 bp deletion at target site of gRNA 4 in one sample Supplemental Table 1 – Primers used in this study Supplemental Table 2 – Comparison of editing frequency from subcloning and deconvolution Appendix S1 – DNA sequence of the vector (CPGE_VEC00447) Acknowledgements The Deutsche Forschungsgemeinschaft supported this work under Germany’s Excellence Strategy – EXC-2048/1 (project ID 390686111), and STE 2654/4. Footnotes Email addresses: suvol101{at}hhu.de , m.stetter{at}uni-koeln.de , henselg{at}hhu.de References ↵ Castellanos-Arévalo , A.P. , Estrada-Luna , A.A. , Cabrera-Ponce , J.L. , Valencia-Lozano , E. , Herrera-Ubaldo , H. , de Folter , S. , et al. ( 2020 ) Agrobacterium rhizogenes-mediated transformation of grain (Amaranthus hypochondriacus) and leafy (A. hybridus) amaranths . Plant Cell Rep , 39 , 1143 – 1160 . OpenUrl CrossRef PubMed ↵ Fauser , F. , Schiml , S. , and Puchta , H. ( 2014 ) Both CRISPR / C as-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana . The Plant Journal , 79 , 348 – 359 . OpenUrl CrossRef PubMed Web of Science ↵ He , Y. , Zhang , T. , Sun , H. , Zhan , H. , and Zhao , Y. ( 2020 ) A reporter for noninvasively monitoring gene expression and plant transformation . Hortic Res , 7 , 152 . OpenUrl CrossRef PubMed ↵ Hoffie , I.V.O. ( 2022 ) Entwicklung des modularen CasCADE-Vektorsystems und dessen Verwendung zur gezielten Mutagenese der Stp13-Orthologe von Weizen und Gerste für die Etablierung dauerhafter Resistenz gegen Rost-und Mehltaupilze . ↵ Joshi , D.C. , Sood , S. , Hosahatti , R. , Kant , L. , Pattanayak , A. , Kumar , A. , et al. ( 2018 ) From zero to hero: the past, present and future of grain amaranth breeding . Theor Appl Genet , 131 , 1807 – 1823 . OpenUrl CrossRef PubMed ↵ Scheben , A. , Wolter , F. , Batley , J. , Puchta , H. , and Edwards , D. ( 2017 ) Towards CRISPR /Cas crops – bringing together genomics and genome editing . New Phytologist , 216 , 682 – 698 . OpenUrl CrossRef PubMed ↵ Shan , S. , Soltis , P.S. , Soltis , D.E. , and Yang , B. ( 2020 ) Considerations in adapting CRISPR/Cas9 in nongenetic model plant systems . Appl Plant Sci , 8 , e11314 . OpenUrl CrossRef ↵ Timoneda , A. , Feng , T. , Sheehan , H. , Walker-Hale , N. , Pucker , B. , Lopez-Nieves , S. , et al. ( 2019 ) The evolution of betalain biosynthesis in Caryophyllales . New Phytol , 224 , 71 – 85 . OpenUrl CrossRef PubMed ↵ Winkler , T.S. , Vollmer , S.K. , Dyballa-Rukes , N. , Metzger , S. , and Stetter , M.G. ( 2024 ) Isoform-resolved genome annotation enables mapping of tissue-specific betalain regulation in amaranth . New Phytologist , nph.19736 . ↵ Ye , C.-Y. and Fan , L. ( 2021 ) Orphan Crops and their Wild Relatives in the Genomic Era . Molecular Plant , 14 , 27 – 39 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted February 25, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following First Successful Targeted Mutagenesis Using CRISPR/Cas9 in Stably Transformed Grain Amaranth Tissue Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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