Background: The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition. Results: In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype. Conclusions: The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high β-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.

With the advent of the new genome editing tool of target-specifically customizable endonucleases, a huge variety of novel opportunities have become feasible. The crop improvement is one of the main applications of genome editing in plant science and plant biotechnology. The amount of publications referring to genome editing and CRISPR/Cas system based molecular tools application in crops is permanently growing. The aim of this study is the systematization and cataloging of these data. Earlier we published the first catalog of targeted crop genome modifications as of February 10, 2017. The current review is an update of the catalog; it covers research papers on crop genome modifications from February 10, 2017 to August 17, 2018, found by searching 47 crop names in the Scopus database. Over one year and a half, 377 articles mentioning CRISPR/Cas and crop names have been published, of which 131 articles describe an experimental application of this tool for editing 193 genes in 19 crops, including rice with the largest number of genes modified (109 genes). Editing 50 of 193 genes was aimed at crop improvement. The catalog presented here includes these 50 genes, specifying the cultivars, each gene and gene product function, modification type and delivery method used. The current full list of genes modified with CRISPR/Cas with the aim of crop improvement is 81 in 16 crops (for 5 years from August 2013 to August 2018). In this paper, we also summarize data on different modifications types in different crops and provide a brief review of some novel methods and approaches that have appeared in crop genome editing research over the reviewed period. Taken together, these data provide a clear view on current progress in crop genome modifications and traits improvement using CRISPR/Cas based genome editing technology.

The CRISPR/Cas system is one of the most promising genome editing tools. It can provide the development of modified nontransgenic plants with the possibility of simultaneous multiple targeted mutation

Blue barley grain pigmentation results from anthocyanin accumulation in the aleurone layer. Anthocyanins are known for their beneficial effects on human health. The gene encoding the MYELOCYTOMATOSIS 2 (MYC2) transcription factor is potentially responsible for the blue coloration of the aleurone. In non-pigmented barley, a single nucleotide insertion in this gene causes a frameshift mutation with a premature stop codon. It was hypothesized that restoring the MYC2 reading frame could activate anthocyanin accumulation in the aleurone. Using a targeted mutagenesis approach in the present study, the reading frame of MYC2 was restored in the non-pigmented cultivar Golden Promise. Genetic constructs harboring cas9 and gRNA expression units were developed, pre-validated in protoplasts, and then functional MYC2 alleles were generated at the plant level via Agrobacterium-mediated transformation. Anthocyanin accumulation in the aleurone layer of grains from these mutants was confirmed through microscopy and chemical analysis. The expression of anthocyanin biosynthesis genes was analyzed, revealing that the restoration of MYC2 led to increased transcript levels of F3H and ANS genes. These results confirm the critical role of the MYC2 transcription factor in the blue aleurone trait and provide a biotechnological solution for enriching barley grain with anthocyanins.

Plant surface properties are crucial determinants of resilience to abiotic and biotic stresses. The outer layer of the plant cuticle consists of chemically diverse epicuticular waxes. The WAX INDUCER1/SHINE subfamily of APETALA2/ETHYLENE RESPONSIVE FACTORS regulates cuticle properties in plants. In this study, four barley genes homologous to the Arabidopsis thaliana AtWIN1 gene were mutated using RNA-guided Cas9 endonuclease. Mutations in one of them, the HvWIN1 gene, caused a recessive glossy sheath phenotype associated with β-diketone deficiency. A complementation test for win1 knockout (KO) and cer-x mutants showed that Cer-X and WIN1 are allelic variants of the same genomic locus. A comparison of the transcriptome from leaf sheaths of win1 KO and wild-type plants revealed a specific and strong downregulation of a large gene cluster residing at the previously known Cer-cqu locus. Our findings allowed us to postulate that the WIN1 transcription factor in barley is a master mediator of the β-diketone biosynthesis pathway acting through developmental stage- and organ-specific transactivation of the Cer-cqu gene cluster.

Система CRISPR/Cas – один из самых перспективных способов геномного редактирования. Этот доступный метод позволяет получать нетрансгенные растения с заданными модификациями, причем можно одновременно производить мутации в нескольких мишенях. Цель настоящего обзора – анализ опубликованных работ, в которых система CRISPR/Cas использована для модификации генов сельскохозяйственных растений, с тем чтобы оценить потенциал этой технологии как нового метода селекции растений. Для 45 сельскохозяйственных культур проведен поиск по сочетанию ключевого слова CRISPR с названием культуры (поиск осуществлялся в названиях, аннотациях и ключевых словах статей из журналов, индексируемых в базе данных Scopus). Среди 206 результатов поиска только 88 содержали описание экспериментальных работ, в которых использована система CRISPR/Cas. В этих работах описаны 145 генов-мишеней у 15 сельскохозяйственных культур, включая рис, у которого модифицировано наибольшее число генов – 78. Возможность получения модифицированных нетрансгенных растений продемонстрирована в большинстве работ. Однако в основном исследования были нацелены на апробацию метода или на изучение функций целевых генов, и лишь редактирование 37 генов связано с улучшением свойств растений. В обзоре представлена таблица-каталог данных генов. Основной используемый вариант модификации – нокаут генов, преимущественно негативных регуляторов роста и развития растений или факторов, определяющих чувствительность к патогенам. В большинстве случаев проверен фенотип модифицированных растений и показано наличие заданных изменений признаков. Однако ввиду того, что негативные регуляторы – это ограниченная группа генов растений, можно предположить, что CRISPR/Cas-направленный нокаут как способ улучшения сельскохозяйственных культур имеет определенные рамки. В связи с этим целесообразно расширение апробации CRISPR/Cas для получения более сложных модификаций в геномах культурных растений, таких как замена дефектных аллелей функциональными или вставка в геном целевых генов (в настоящее время для улучшения свойств сельскохозяйственных растений известны лишь единичные примеры таких модифика- ций). Другое важное условие для широкого практического использования системы геномного редактирования CRISPR/Cas в селекции – возможность применения ко многим сортам одного и того же вида. В опубликованных работах пока еще используется весьма ограниченное число модельных сортов/линий. Тем не менее, несмотря на описанные ограничения, необходимо подчеркнуть, что за короткий срок (3.5 года с момента опубликования первых работ по модификации генома растений с помощью системы CRISPR/Cas) достигнуты значительные успехи.