A highly efficient CRISPR-Cas9-based gene editing system in oat (Avena sativa)

A highly efficient CRISPR-Cas9-based gene editing system in oat (Avena sativa) | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A highly efficient CRISPR-Cas9-based gene editing system in oat ( Avena sativa ) View ORCID Profile Mehtab-Singh , Cali Kaye , View ORCID Profile Rajvinder Kaur , View ORCID Profile Jaswinder Singh doi: https://doi.org/10.1101/2025.01.26.633040 Mehtab-Singh 1 Plant Science Department, 21111 Rue Lakeshore, McGill University , Montreal, QC H9X 3V9, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mehtab-Singh Cali Kaye 1 Plant Science Department, 21111 Rue Lakeshore, McGill University , Montreal, QC H9X 3V9, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rajvinder Kaur 1 Plant Science Department, 21111 Rue Lakeshore, McGill University , Montreal, QC H9X 3V9, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rajvinder Kaur Jaswinder Singh 1 Plant Science Department, 21111 Rue Lakeshore, McGill University , Montreal, QC H9X 3V9, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jaswinder Singh For correspondence: jaswinder.singh{at}mcgill.ca Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Cultivated oat ( Avena sativa ) is an emerging cereal for healthy lives owing to its unique characteristics, such as high β-glucan and oil content, distinctive fatty acid composition, and gluten-free nature. The recent unravelling of the 12.5 Gb hexaploid oat genome underlined breeding barriers caused by ancestral translocations and inversions, leading to recombination suppression and pseudo-linkage further hindering conventional trait introgression. Over the past decade, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system has been extensively used for crop improvement and functional genomics in all other cereals except oats. Its large repetitive genome with three sub-genomes, lack of efficient transformation, recalcitrant nature, and complex molecular screening due to gene redundancy have been major obstacles to gene editing success in oat. We report the first successful CRISPR-Cas9-based gene editing in oat in three genes — AsTLP8, AsVRN3 and AsVRN3D with gene-editing efficiency of up to 41.1%. The gene-edited plants for all the genes carried deletions and/or one base insertion. Further analysis of VRN3 T 1 and T 2 mutants revealed bent leaves in heterozygous knockouts (AACCdD), while an extended vegetative growth phase was seen in the T 1 homozygous and biallelic mutants (aaccdd), accentuating the important role of VRN3 in oat development. We are confident that this highly efficient oat gene editing system will pave the way for a deeper molecular understanding of this healthy cereal, deciphering oat’s functional genomics, and creating genetic diversity at the cold spots of recombination in oat. Cultivated oat ( Avena sativa ), a cereal of worldwide importance, belongs to the Aveneae family of cereals, distinguishing it from wheat, rye, barley, and rice. This distinction explains its unique characteristics, such as high β-glucan and oil content, distinctive fatty acid composition, and gluten-free nature, making it suitable for both human consumption and animal feed. Since the 1870s, oat breeding programs have achieved remarkable improvements in various traits, including disease resistance, yield, milling quality, and β-glucan content. However, the process remains lengthy, labour-intensive, and cumbersome. The recent unravelling of the massive 12.5 Gb oat genome revealed breeding barriers owing to the ancestral large-scale translocations and inversions ( Kamal et al., 2022 ). Hence, the traditional introgression of traits through breeding cannot be attained due to recombination suppression and pseudo-linkage at such genetic loci. This highlights the urgent need to employ the latest genome editing technologies for oat improvement, which holds the utmost potential for altering specific gene functions and introducing allelic diversity. Over the past decade, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- Cas9 system has been extensively used for crop improvement and functional genomics in all other cereals except oats ( Ahmar et al., 2024 ). Its large repetitive genome with three sub-genomes, lack of efficient transformation, recalcitrant nature, and complex molecular screening due to gene redundancy are major obstacles to gene editing success. We report the first successful CRISPR-Cas9-based gene editing in oat, generating deletions and insertions. To test whether the CRISPR-Cas9 system can produce targeted gene editing in oat, different single guide RNAs (sgRNAs) were designed to target the Thaumatin-like protein 8 (TLP8), Vernalization 3 (VRN3) , and Vernalization 3-D (VRN3D) in hexaploid oat ( Figure 1a, b and Figure S5). TLP8 has been associated with β-glucan the primary heart-healthy fibre that makes oats valuable for human consumption ( Singh et al., 2017 ). Concomitantly, VRN3 is a member of the PEBP (Phosphatidylethanolamine-binding protein) family and has been mapped to a QTL associated with plant height, oil content and other crucial yield-related traits in oat. Intriguingly, VRN3D is in a recombination-suppressed region on the inverted 7D chromosome, making it an interesting candidate for gene editing ( Tinker et al., 2022 ). Mature seeds of the hexaploid spring oat variety Park were selected for transformation due to the recent success in our lab with the introduction of Ac/Ds elements and the refinement of fatty acid composition using particle gun bombardment ( Mahmoud et al., 2022 ; Zhou et al., 2024 ). Seeds were sterilized with bleach, and calli were produced for plant transformation. The transformed calli underwent three rounds of hygromycin selection (20mg/L) followed by regeneration and rooting of transgenic calli on the respective media supplemented with 5mg/L hygromycin ( Figure 1h ; Methods S1). Three individual constructs (pJDTLP8, pJDVRN3, and pJDVRN3D) were designed by cloning the gene-specific guides driven by wheat U6 promoter in the JD633 backbone with ubiquitin promoters for GRF-GIF chimera, Cas9 , and hygromycin (hpt) ( Debernardi et al., 2020 ) ( Figure 1a,b and Figure S5). For the TLP8 gene, a total of 100 calli were bombarded with the pJDTLP8 construct, producing 21 transgenic plants with a transformation efficiency of 21% (Figure S1). The guide flanking region of the AsTLP8 was then amplified using TLP8 gene primers (Table S1) and subjected to next-generation sequencing (NGS). The analysis confirmed seven T 0 gene-edited plants reporting a highly efficient gene editing frequency of 41.1% ( Figure 1f ). Notably, the PCK-7 plant had a 4bp deletion in the TLP8C , an 11bp deletion and a 1bp (T) insertion in the TLP8D ( Figure 1c ). Download figure Open in new tab Figure 1. Targeted CRISPR/Cas9-mediated gene-editing in oat. (a) CRISPR construct pJDTLP8 with TLP8 gRNA from the conserved region. (b) CRISPR construct pJDVRN3 with VRN3 gRNA from the conserved region. PAM is depicted in red. (c) Targeted mutagenesis in the TLP8 guide region. Deletions are depicted with a dashed line and insertion with a red box leading to a frameshift mutation. Mutation types are indicated in red on the right. (d) Targeted mutagenesis in the VRN3 guide region. Deletions are shown with a dashed line and insertion with a red box leading to a frameshift mutation. Mutation types are indicated in red on the right. (e) PCR screening with hpt gene primers in pJDVRN3 transformed lines. WT: non-transformed control plant; NC: negative control without DNA. (f) Summary of transformation frequency, gene-editing efficiency and transgenerational inheritance. (g) Cleaved amplified polymorphic sequence (CAPS) assay with NcoI for screening of gene-edited pJDVRN3 T 0 plants. The red star indicates an undigested amplicon emerged due to gene editing. (h) Hygromycin selection (20mg/L) of transgenic calli. (i) Maximum intensity projected para-dermal view of WT and T 1 line #25F-17 flag leaf segments stained with propidium iodide. Bars = 50 μm. (j) Phenotyping of VRN3 single copy knockout T 1 mutants. Bend flag leaves in the heterozygous line #25F-17 are magnified on the top, while for #25F-10, they are indicated with red arrows. Scale bar = 7 cm. (k) Phenotyping of VRN3 triple knockout T 1 mutants. Scale bar = 12 cm. (l) Zygosity confirmation of VRN3 gene-edited T 1 plants via NGS. Deletions are shown with a dashed line and insertions with a red box. Base pair alterations are indicated in red on the right. In another experiment, 75 calli were bombarded with pJDVRN3 targeting the oat VRN3 , yielding six transgenic plants confirmed through hygromycin (hpt) gene PCR with a mean transformation efficiency of 8% ( Figure 1e,f ; Table S1). All the plants were successfully regenerated and transferred to soil in the growth chamber. The target region was PCR amplified from the T 0 transgenic lines using the VRN3 primers (Table S1) and sequenced by NGS. The sequencing results reported gene-edited plants with small deletions and insertions in the VRN3 gene ( Figure 1d ). Intriguingly, the 4bp deletion in VRN3D altered the NcoI restriction site that facilitated the screening of knockout mutants through cleaved amplified polymorphic sequence (CAPS) assay. The CAPS genotyping identified the gene-edited lines with 4bp deletion depicting undigested mutated PCR amplicon, while the WT control was completely digested ( Figure 1g ). The undigested amplicon was gel extracted and 4bp deletion was confirmed by Sanger sequencing (Figure S4). In total, three gene-edited plants were obtained in the T 0 generation, with mutations in all three VRN3 copies reporting a high gene editing efficiency of 50 % ( Figures 1d,f ). Since we were anticipating a perceptible phenotype in the VRN3 lines, they were advanced to the next generations. To test the heritability and transgene segregation, 65 T 1 plants of the JMV3-25F T 0 line were screened at the guide region using the CAPS assay, followed by NGS and Sanger sequencing. DNA was amplified from 65 T 1 lines using VRN3D-specific gene primers followed by restriction digestion with NcoI as the restriction site has been altered in VRN3D -edited plants (Table S1). Of the 65 T 1 plants screened using CAPS assay, 51 78.5%) contained gene edits for VRN3D ( Figure 1f and Figure S2). Further analysis revealed 38 Cas9-free plants, out of which two were homozygous, while 26 plants had heterozygous mutations ( Figure 1f and Figure S3). The plants were phenotyped in a controlled growth chamber under long-day conditions (16h light/8h dark). Two distinct and intriguing phenotypes were observed in the T 1 progeny, and their genotypes were extrapolated through NGS using homoeologous gene-specific primers. One set of plants (#25-17, #25F-10, and #25F-39) displayed impaired flag leaf development (sharp bend near the flag leaf tip) at the booting stage (Z41) as compared to the WT and null plants that showed healthy upright flag leaves ( Figure 1j ) ( Zadoks et al., 1974 ). Such plants carried a heterozygous mutation in the VRN3D , while the VRN3A and VRN3C remained unedited (AACCdD) ( Figure 1l , Figure S2, S4). Transgenerational inheritance of the bent leaf trait was consistently observed in the T 2 generation of the #25F-17 plant. This phenotype was further validated in other VRN3D mutants generated through a VRN3D -specific guide (Figure S5). The bend site was further investigated under a confocal microscope, revealing changes in the tissue arrangement and morphology of epidermal cells in the mutant compared to the wild type ( Figure 1i ). Since VRN genes belong to the PEBP family, we speculate that the PEBP protein may be involved in controlling plant epidermal cell patterning and differentiation, as observed in other organisms ( Trakul et al., 2005 ). On the contrary, several plants exhibited only a vegetative growth phase and were genotypically characterized as triple knockouts with biallelic or homozygous mutations in all VRN3 copies (aaccdd) ( Figure 1k,l ). However, it remains to be seen whether the conserved role of the SPL /miR156 module in inflorescence development and reproductive phase transition in oat has been affected (Mehtab-Singh et al., 2024). In summary, we report the first successful CRISPR-Cas9-based gene editing in oat in three different genes — AsTLP8, AsVRN3 and AsVRN3D with high gene-editing efficiencies. The gene-edited plants for all the genes carried deletions and/or one base insertion. Analysis of VRN3 mutant T 1 and T 2 plants revealed bent leaves in single-copy knockouts (AACCdD), while an extended vegetative growth phase was seen in the T 1 triple-knockout mutants (aaccdd), accentuating the important role of VRN3 in oat development. We are confident that this highly efficient oat gene editing system will pave the way for a deeper molecular understanding of this healthy cereal, deciphering oat’s functional genomics, and creating genetic diversity at the cold spots of recombination in oat. Author contributions JS and M-S designed the experiments. M-S and CK designed the gRNA and constructs. M-S, CK and RK performed the oat transformations and tissue culture. M-S and CK conducted the molecular and phenotypic screening. M-S made the figures and wrote the manuscript, and all authors revised it. Conflict of interest The authors declare no competing interests. Supporting Information Table S1 List of primers used in the study. Methods S1 Material and Methods Figure S1 Confirmation of transgenic lines in pJDTLP8 experiment. Figure S2 CAPS assay on T 1 transgenic lines from JMV3-25F. Figure S3 Screening of Cas9-free plants in the JMV3-25F T 1 generation. Figure S4 Sanger sequencing of VRN3 mutant lines. Figure S5 Targeted gene editing in the VRN3D gene. Acknowledgments This study was financially supported by the Prairie Oat Growers Association (POGA) through Agriculture Funding Consortium. We also acknowledge the support of the NSERC-CREATE program on Genome Editing for Food Security and Environmental Sustainability (GEFSES). We sincerely thank McGill University ECP3-Multi-Scale Imaging Facility, Sainte-Anne-de-Bellevue, Canada and especially Diksha Bhola (McGill University, Montreal) for her assistance with sample preparation and confocal microscopy imaging. References ↵ Ahmar , S. , Usman , B. , Hensel , G. , Jung , K.H. , Gruszka , D. , 2024 . CRISPR enables sustainable cereal production for a greener future . Trends Plant Sci . doi: 10.1016/j.tplants.2023.10.016 OpenUrl CrossRef PubMed ↵ Debernardi , J.M. , Tricoli , D.M. , Ercoli , M.F. , Hayta , S. , Ronald , P. , Palatnik , J.F. , Dubcovsky , J. , 2020 . A GRF–GIF chimeric protein improves the regeneration efficiency of transgenic plants . Nat Biotechnol 38 , 1274 – 1279 . doi: 10.1038/s41587-020-0703-0 OpenUrl CrossRef PubMed ↵ Kamal , N. , Tsardakas Renhuldt , N. , Bentzer , J. , Gundlach , H. , Haberer , G. , Juhász , A. , Lux , T. , Bose , U. , Tye-Din , J.A. , Lang , D. , van Gessel , N. , Reski , R. , Fu , Y.B. , Spégel , P. , Ceplitis , A. , Himmelbach , A. , Waters , A.J. , Bekele , W.A. , Colgrave , M.L. , Hansson , M. , Stein , N. , Mayer , K.F.X. , Jellen , E.N. , Maughan , P.J. , Tinker , N.A. , Mascher , M. , Olsson , O. , Spannagl , M. , Sirijovski , N. , 2022 . The mosaic oat genome gives insights into a uniquely healthy cereal crop . Nature 606 , 113 – 119 . doi: 10.1038/s41586-022-04732-y OpenUrl CrossRef PubMed ↵ Mahmoud , M. , Zhou , Z. , Kaur , R. , Bekele , W. , Tinker , N.A. , Singh , J. , 2022 . Toward the development of Ac/Ds transposon-mediated gene tagging system for functional genomics in oat (Avena sativa L .). Funct Integr Genomics 22 , 669 – 681 . doi: 10.1007/s10142-022-00861-9 OpenUrl CrossRef PubMed Mehtab-Singh Tripathi , R.K. , Bekele , W.A. , Tinker , N.A. , Singh , J. , 2024 . Differential expression and global analysis of miR156/SQUAMOSA promoter binding-like proteins (SPL) module in oat . Scientific Reports 2024 14:1 14 , 1 – 13 . doi: 10.1038/s41598-024-60739-7 OpenUrl CrossRef PubMed ↵ Singh , S. , Tripathi , R.K. , Lemaux , P.G. , Buchanan , B.B. , Singh , J. , 2017 . Redox-dependent interaction between thaumatin-like protein and β-glucan influences malting quality of barley . Proc Natl Acad Sci U S A 114 , 7725 – 7730 . doi: 10.1073/PNAS.1701824114/SUPPL_FILE/PNAS.201701824SI.PDF OpenUrl Abstract / FREE Full Text ↵ Tinker , N.A. , Wight , C.P. , Bekele , W.A. , Yan , W. , Jellen , E.N. , Renhuldt , N.T. , Sirijovski , N. , Lux , T. , Spannagl , M. , Mascher , M. , 2022 . Genome analysis in Avena sativa reveals hidden breeding barriers and opportunities for oat improvement . Communications Biology 2022 5:1 5 , 1 – 11 . doi: 10.1038/s42003-022-03256-5 OpenUrl CrossRef PubMed ↵ Trakul , N. , Menard , R.E. , Schade , G.R. , Qian , Z. , Rosner , M.R. , 2005 . Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation . J Biol Chem 280 , 24931 – 24940 . doi: 10.1074/JBC.M413929200 OpenUrl Abstract / FREE Full Text ↵ Zadoks , J.C. , Chang , T.T. , Konzak , C.F. , 1974 . A decimal code for the growth stages of cereals . Weed Res 14 , 415 – 421 . doi: 10.1111/J.1365-3180.1974.TB01084.X OpenUrl CrossRef ↵ Zhou , Z. , Kaur , R. , Donoso , T. , Ohm , J-B. , Gupta , R. , Lefsrud ., M. , Singh , J. , 2024 . Metabolic Engineering-Induced Transcriptome Reprogramming of Lipid Biosynthesis Enhances Oil Composition in Oat . Plant Biotechnology Journal , DOI: 10.1111/pbi.14467 . OpenUrl CrossRef View the discussion thread. Back to top Previous Next Posted January 28, 2025. 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Share A highly efficient CRISPR-Cas9-based gene editing system in oat ( Avena sativa ) Mehtab-Singh , Cali Kaye , Rajvinder Kaur , Jaswinder Singh bioRxiv 2025.01.26.633040; doi: https://doi.org/10.1101/2025.01.26.633040 Share This Article: Copy Citation Tools A highly efficient CRISPR-Cas9-based gene editing system in oat ( Avena sativa ) Mehtab-Singh , Cali Kaye , Rajvinder Kaur , Jaswinder Singh bioRxiv 2025.01.26.633040; doi: https://doi.org/10.1101/2025.01.26.633040 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41909) Biophysics (21435) Cancer Biology (18576) Cell Biology (25479) Clinical Trials (138) Developmental Biology (13366) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24301) Genetics (15598) Genomics (22482) Immunology (17726) Microbiology (40359) Molecular Biology (17162) Neuroscience (88529) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4820) Physiology (7636) Plant Biology (15125) Scientific Communication and Education (2044) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)