Frontiers in Genetics, Journal Year: 2024, Volume and Issue: 15

Published: Dec. 16, 2024

Hazelnuts are trees belonging to the Betulaceae family and Corylus genus (Wani et al., 2020). Due their delicious flavor profile, nutrient composition, antioxidant properties, hazelnuts widely used as whole nuts or processed foods. highly appreciated popular species cultivated across globe, including C. avellana, in Europe; americana, predominantly found North America; heterophylla mandshurica, extensively utilized Asia (Botta 2019). Thus far, several pathogens such Xanthomonas sp., Pseudomonas Botrytis cinerea, Alternaria Cytospora sp.Phytophthora sp. various pests compromise nut production (Guerrero 2014;Battilani 2018;Sun 2023), which also constantly faces environmental stress (Allegrini 2022).Plants engaged a struggle for survival adaptation, conventional breeding techniques have allowed development of hazelnut cultivars with improved characteristics especially related cold resistance yield (Wang 2018a;Botta 2019;Mehlenbacher Molnar, 2021). However, efficiency classical approaches depends on availability genomic resources may be limited commercial varieties due introduction undesired genetic traits during steps. Furthermore, approach has been recognized time-consuming process that requires multiple generations years introduce fix desirable (Tester Langridge, 2010). Significant support research improvement come from new genome editing revolutionizing plant programs functional studies. In particular, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 technology successfully applied fruit nuts, becoming technique enhancing biotic abiotic tolerance plants 2018b;Chang 2022). The CRISPR-Cas9 system employs Cas9 nuclease, able induce DNA double-strand breaks (DSB) (Puchta, 2017). Once DSB is created, cell's natural repair mechanisms into play, leading non-homologous end joining (NHEJ) homology-directed (HDR). NHEJ often leads insertions deletions (indels) at break site, can result gene knockout, while HDR precise edits when donor template provided (Khan 2018). guidance these modifications by guide RNAs (gRNAs) designed effectively nuclease intended target sites (Hsu 2013). success heavily relies design specific gRNAs (Filippova For example, (Evangelista 2024) suggested targeting domains allergenic genes could reduce unintended effects caused complete silencing. This would enhance hypo-allergenicity without compromising fitness (Tran Indeed, this strategy employed previous studies where enzyme was directed toward associated susceptibility 2021).Thereby, defined simple, efficient, specific, cost-effective method facilitate generation transgenefree edited shorter period compared breeding.Numerous web-based tools developed variety species. Available online software selecting optimal sgRNA targets based user-defined parameters (Uniyal 2019;Labun 2019;Haeussler 2016;Liu 2017;Bae 2014). date, user-friendly integrating genomes not yet available, presents significant gap researchers area.Crucial application came recent released assemblies different species, providing insights diversity evolutionary relationships (Li 2021;Lucas 2021;Zhao 2021;Brainard 2024). High-quality sequences curated prediction essential identifying suitable gRNA (Mohr 2016). factors influence effectiveness, efficiency, uniqueness genes, matching gene, position Protospacer Adjacent Motif (PAM) sequence, accessibility within chromatin structure (Jensen 2017), formation secondary structures (Riesenberg it shown self-folding free energy strongly influences cleavage Therefore, activity predicted methods off-target scores evaluating potential cutting loci 2016).However, currently available do allow determining (Hassan Guides assessing through (Bae 2014b;Montague 2014;Moreno-Mateos 2015;Chuai 2018;Concordet Haeussler, 2018;Cui Algorithms Rule Set 1and 2 on-target (Gagnon 2014;Heigwer 2014;Wang 2014;Xu 2015). These algorithms take account features like nucleotide GC content, positional forecast efficacy objective maximizing activity. Conversely, predicting effects, CFD (Cutting Frequency Determination), Mismatch count, MIT specificity (Cong 2013;Hsu 2013;Mali 2013;Doench 2014Doench , 2016)). employ scoring systems mismatches sequence anticipate gRNAs. Recent pointed out reliability accuracy score Count applications (Liu 2020;Naeem dedicated databases (DB) real molecular biologists programs. lackedCorylus reference genomes, bioinformatics custom analysis advanced command-line skills. limitation made difficult access simple intuitive interfaces designing Additionally, require identification duplicated (paralogs). Plant frequently host groups evolved common ancestor retaining overlapping redundant functions. poses challenge genetics makes crucial step (Bhuyan 2023).Therefore, an atlas selection simultaneous silencing utilizing Homologs Direct Repair (Aksoy view, comprehensive DB containing all information represents advantage one most critical steps application.To end, we single multi-target database (SiMul-db)  Paralog will multi-copy targets. Orthology inference permit transfer function model toCorylus genes.To develop plants, European (Corylus avellana) 'Tombul' (v2.4) its annotation reported Lucas al. (2021) To obtain whole-genome libraries (RD)-build implemented CRISPR-Local using -U 15 -D 3 settings (Sun (.fa) corresponding annotations (.gff) heterophylla, mandshurica were input files. screening possible algorithm (Doench While each site highest frequency determination (CFD) gRNA, realized SeqMap program (Jiang Wong, 2008). All data determined entire exported RD format (Supplementary Tables 1234), includes about physical position, relative against transcription start score, every locus. Database (DB)search sorted results annotated Cor genes. Paralogs (PL)-search extract multi-gene targets.To provide deep understanding evolution diversification OrthoFinder v2.5.1 package (Emms Kelly, Simultaneously, avellana A. thaliana proteomes analyzed, default settings. package, BLAST tool fast similarity searches among protein sequences. clustering inferred MCL algorithm; unrooted tree orthogroup DendroBLAST (Kelly domain architecture Pfam InterProScan v5.69-101.0 (Jones 2014) setting.The RNA comparison calculated RNAfold ViennaRNA (version 2.6.4) (Lorenz 2011).Specifically, propensity form calculating selffolding (ΔG expressed kcal/mol) -d2 option dangling-end model, allowing contribute favorable interactions.Over thirteen million four date (Table 1). Future updates SiMul-db incorporate newly sequenced assemblies, further expanding increasing number design. values range 0 1, higher considered perform better 2014a). Considering high obtained gRNAs, selected than 0.66, obtaining subset 1,025,628 top rank 2016;Haeussler Interestingly, 71,262 classified On average, non-functional had significantly ones (Wong hone evaluation, estimated (ΔG) determine structures. Generally, fold itself ΔG value more negative, hinders pairing (Kesavan Nair,2023). According Jensen (2017), ability endonuclease efficiently cleave greater comprised between -2 kcal/mol. our database, ~80% best showed > dataset orthology proteomes, outgroup genome. chosen widespread use extensive knowledge (Cao 2011). 21,237 orthogroups Table 5-6, Supplementary Figure spp. speed up discovery paralogs future (Mota Moreover, predictions tailored interests 78910). Finally, lower guides data, sequences, models, generate (Figure Protein obtaintedconsulting (Pfam) proteome included comparative purposes. Guide Cas9-gRNAs identify homolog groups, respectively estimation additional accurate guides. workflow users (Armario Najera Users choose considering (on target, CFD, ΔG) scores, region coding suggests multi-editing streamlined allows efficient biotechnology assisted genus. Specific agricultural traits, metabolic pathways, responses stresses SiMul-db. vulnerable commonly known "gray mold", fungal pathogen affecting (Romanazzi Feliziani, infect parts hazelnut, fruits, inducing losses quality deterioration assist valuable strategies. Below two strategies involved B. cinerea interaction. Previous AtDND1 (AT5G15410) AtPUB17 (AT1G29340), potentially plantpathogen 2017;Ramirez Gaona 2023). 7). exploring SiMul-db, easily reveal orthologs (OG0011955: CamerRush.05G196000.1, Cav05g20890.1, EVM0018229.1, CmaG0015144.1)to 5), find identified orthologous 11). three Cav02g18830.1, Cav02g18860.1, Cav02g18960.1). By querying simultaneously 12).(SiMul-db emerges innovative accelerating hazelnuts. It provides lists low first time, consolidated unique reduces risk enhances CRISPR-Cas9. Even absence agrobacterium-mediated transformation protocols, consulted alternative methods, transient (Son Park, implementations include other making accessible researchers. studies, ultimately boosting productivity resilience.The authors declare conducted any financial construed conflict interest.

Language: Английский