Sorghum bicolor (L.) Moench (sorghum) is cultivated as food for humans and livestock and is valued for its low input requirements. However, sorghum grain protein is deficient in the essential amino acid lysine and has poor protein digestibility. This is because highly abundant proline-rich kafirins constitute >70% of proteins and form low-digestibility protein bodies. To reduce kafirin expression in the endosperm and elicit wholesale proteome rebalancing to increase non-kafirin proteins, a single guide RNA (sgRNA) Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) construct was previously used to target members of the highly repetitive alpha-kafirin family, k1C, in RTx430 sorghum. The current study was conceived to evaluate the nutritional and biophysical characteristics of two sorghum lines with k1C family deletions compared to unedited RTx430. Despite confirming a ~400-kb deletion in the k1C family of both edited lines encompassing seven active genes, no significant decrease was observed in k1C expression or compensatory increase in non-kafirin proteins. A significant increase was observed in protein-bound amino acids in both k1C-deleted lines and in both edited lines relative to unedited RTx430, which increased total seed protein in one line. Protein bodies were observed to be more irregularly shaped, with seeds retaining wild-type levels of vitreous endosperm. RNA Isoform Sequencing (Iso-Seq) was performed to measure k1C family gene expression and capture differentially expressed non-kafirin genes. Five k1C members were observed to have biologically significant expression, with only two of the residual, non-deleted k1C genes having elevated expression in lines with the deletion. The results of this study demonstrate the ability of CRISPR/Cas9 editing of kafirins to elicit changes to the amino acid profile of sorghum and increase protein. Moreover, the results demonstrate an extreme example of the ability of highly repetitive genome regions such as the k1C subfamily to compensate for the effects of CRISPR-induced multigene deletions.

Inorganic nitrogen (N) fertilizer has emerged as one of the key factors driving increased crop yields in the past several decades; however, the overuse of chemical N fertilizer has led to severe ecological and environmental burdens. Understanding how crops respond to N fertilizer has become a central topic in plant science and plant genetics, with the ultimate goal of enhancing N use efficiency (NUE) in crop production. N, being one of the most essential macronutrients, significantly impacts crop performance across different stages of plant development, as the phenotypic traits result from the accumulative effects of genetic factors, environmental conditions (i.e., N availability), and their interactions. To characterize the N response and growth trajectory, we generated sorghum mutants using CRISPR technology. Using a LemnaTec plant imaging system, we obtained time series imagery data for these CRISPR-edited mutants under high N and low N greenhouse conditions. After imagery data analysis, we extracted a number of morphological and greenness index traits as a proxy of plant growth and N responses. Subsequently, we employed two different methods to model the temporal N-responsive traits, allowing us to estimate seven key parameters from the growth curve. Results revealed that the wildtype and the edited sorghum lines exhibited differences in N responses for several of the key growth-related parameters. The high-throughput N phenotyping pipeline paves the way for a better understanding of the N responses of edited lines in a dynamic manner and sheds light on further improvements in crop NUE. ### Competing Interest Statement J.C.S. has equity interests in Data2Bio, LLC; Dryland Genetics LLC and EnGeniousAg LLC. He is a member of the scientific advisory board of GeneSeek and currently serves as a guest editor for The Plant Cell. The authors declare no other conflicts of interest associated with this work.

Introduction Sorghum is a resilient and widely cultivated grain crop used for feed and food. However, it’s grain is deficient in lysine, an essential amino acid. This is due to the primary seed storage proteins, the alpha-kafirins, lacking lysine. It has been observed that reductions in alpha-kafirin protein results in rebalancing of the seed proteome and a corresponding increase in non-kafirin proteins which leads to an increased lysine content. However, the mechanisms underlying proteome rebalancing are unclear. This study characterizes a previously developed gene edited sorghum line, with deletions at the alpha kafirin locus. Methods A single consensus guide RNA leads to tandem deletion of multiple members of the gene family in addition to the small target site mutations in remaining genes. RNA-seq and ATAC-seq were utilized to identify changes in gene expression and chromatin accessibility in developing kernels in the absence of most alpha-kafirin expression. Results Several differentially accessible chromatin regions and differentially expressed genes were identified. Additionally, several genes upregulated in the edited sorghum line were common with their syntenic orthologues differentially expressed in maize prolamin mutants. ATAC-seq showed enrichment of the binding motif for ZmOPAQUE 11, perhaps indicating the transcription factor’s involvement in the kernel response to reduced prolamins. Discussion Overall, this study provides a resource of genes and chromosomal regions which may be involved in sorghum’s response to reduced seed storage proteins and the process of proteome rebalancing.

Additional file 4. Sequence data for edits in lemon white1 in three Miscanthus species. Description: Aligned DNA sequence; format: FASTA.

Additional file 2. Sequence and location of three gRNAs in the lemon white1 gene. Description: Multiple sequence alignments of lemon white1 orthologs from sorghum and miscanthus and the three gRNAs used to edit the gene; format: FASTA multiple sequence alignment.

Cannabis sativa is a versatile crop that can be cultivated for fiber, seed, or phytochemicals. To take advantage of this versatility and the potential of Cannabis as a feedstock for the bioeconomy, genomics-enabled breeding programs must be strengthened and expanded. This work contributes to the foundation for such by investigating the phytochemistry and genomics of feral Cannabis populations collected from seventeen counties across the climate gradient of Nebraska. Flower tissue from male and female plants (28 total) was studied using (i) gas chromatography-mass spectrometry to assess cannabinoid profiles and (ii) RNA sequencing to determine tran-script abundances. Both male and female flower tissues produced cannabinoids, and, though the compounds were more abundant in female flower tissue, the primary cannabinoid in both was usually cannabidiol. The expression of genes that mediate early steps on the cannabinoid biosynthetic pathway were upregulated in fe -male relative to male flowers, suggesting that female versus male flower tissue cannabinoid abundance may be controlled at least in part at the transcriptional level. DNA sequencing was used to place feral Cannabis plants from Nebraska into a previously described genomic context, revealing that all the plants studied here are much more similar to previously characterized hemp-type Cannabis plants than to drug-type Cannabis plants, at least at the genetic level. This work provides foundational phytochemical knowledge and a large set of high-quality single nucleotide polymorphism markers for future studies of feral Nebraska Cannabis.

Background Miscanthus , a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariflorus and M. sinensis . Use of biotechnology-based procedures to genetically improve Miscanthus , to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites. Results We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum -like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 ( lw1 ) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1 . Embryogenic calli of M. sacchariflorus , M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1 . Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors. Conclusions We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good editing target in species other than Z. mays . The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop. Graphical Abstract

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Summary Drought stress is the major limiting factor in agriculture. Wheat, which is the most widely grown crop in the world, is predominantly cultivated in drought‐prone rainfed environments. Since roots play a critical role in water uptake, root response to water limitations is an important component for enhancing wheat adaptation. In an effort to discover novel genetic sources for improving wheat adaptation, we characterized a wheat translocation line with a chromosomal segment from Agropyron elongatum, a wild relative of wheat, which unlike common wheat maintains root growth under limited‐water conditions. By exploring the root transcriptome data, we found that reduced transcript level of LATERAL ROOT DENSITY (LRD) gene under limited water in the Agropyron translocation line confers it the ability to maintain root growth. The Agropyron allele of LRD is down‐regulated in response to water limitation in contrast with the wheat LRD allele, which is up‐regulated by water deficit stress. Suppression of LRD expression in wheat RNAi plants confers the ability to maintain root growth under water limitation. We show that exogenous gibberellic acid (GA) promotes lateral root growth and present evidence for the role of GA in mediating the differential regulation of LRD between the common wheat and the Agropyron alleles under water stress. Suppression of LRD also had a positive pleiotropic effect on grain size and number under optimal growth conditions. Collectively, our findings suggest that LRD can be potentially useful for improving wheat response to water stress and altering yield components.

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Synthetic taurine is an important animal feed supplement critical for fish and cat nutrition. However, there is a demand for natural and renewable sources of this nutraceutical additive. The objective of this study was to increase the production of taurine using genetically engineered Chlamydomonas reinhardtii modified to have the cysteine dioxygenase/cysteine sulfinic acid decarboxylase (CDO/CSAD) taurine synthesis pathway from animals. Because unicellular green microalgae produce taurine at low levels, methods to increase taurine content may qualify the use of these microbes as a feed supplement. Here, increased microalgal taurine content was accomplished using a synthetic approach based on addition of the CDO/CSAD taurine biosynthetic pathway from animals into the chloroplasts of Chlamydomonas reinhardtii. To optimize synthesis, genes were codon optimized and transformed as either separate genes (cdo::csad) or as a fusion protein (cdo-csad). Genotyping confirmed the identity of stable homoplasmic cdo::sad and cdo-csad transformants while HPLC-MS was used to identify pathway intermediates and the end product taurine. While the cdo-csad fusion transformants produced only the first taurine intermediate, cysteine sulfinic acid (CSA), the cdo::csad transformants produced CSA, hypotaurine, and taurine indicating successful synthetic expression of the animal taurine pathway in microalgae. Unsuccessful expression of the fusion construct explained the lack of end product formation and resulted from premature transcript cleavage as indicated by Northern and Western blot analysis. Furthermore, culture supplementation with the pathway precursors, cysteine and methionine, improved production of CDO/CSAD pathway intermediates. These results support the feasibility of using engineered microalgae coupled with culture supplementation to meet taurine requirements for use as an animal feed supplement.

To increase phenylpropanoid constituents and energy content in the versatile C4 grass sorghum (Sorghum bicolor [L.] Moench), sorghum genes for proteins related to monolignol biosynthesis were overexpressed: SbMyb60 (transcriptional activator), SbPAL (phenylalanine ammonia lyase), SbCCoAOMT (caffeoyl coenzyme A [CoA] 3-O-methyltransferase), Bmr2 (4-coumarate:CoA ligase), and SbC3H (coumaroyl shikimate 3-hydroxylase). Overexpression lines were evaluated for responses to stalk pathogens under greenhouse and field conditions. Greenhouse-grown plants were inoculated with Fusarium thapsinum (Fusarium stalk rot) and Macrophomina phaseolina (charcoal rot), which cause yield-reducing diseases. F. thapsinum-inoculated overexpression plants had mean lesion lengths not significantly different than wild-type, except for significantly smaller lesions on two of three SbMyb60 and one of two SbCCoAOMT lines. M. phaseolina-inoculated overexpression lines had lesions not significantly different from wild-type except one SbPAL line (of two lines studied) with mean lesion lengths significantly larger. Field-grown SbMyb60 and SbCCoAOMT overexpression plants were inoculated with F. thapsinum. Mean lesions of SbMyb60 lines were similar to wild-type, but one SbCCoAOMT had larger lesions, whereas the other line was not significantly different than wild-type. Because overexpression of SbMyb60, Bmr2, or SbC3H may not render sorghum more susceptible to stalk rots, these lines may provide sources for development of sorghum with increased phenylpropanoid concentrations.

Kafirins are the major storage proteins in sorghum (Sorghum bicolor) grains and form protein bodies with poor digestibility. Since kafirins are devoid of the essential amino acid lysine, they also impart poor protein quality to the kernel. The alpha-kafirins, which make up most of the total kafirins, are largely encoded by the k1C family of highly similar genes. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to target the k1C genes to create variants with reduced kafirin levels and improved protein quality and digestibility. A single guide RNA was designed to introduce mutations in a conserved region encoding the endoplasmic reticulum signal peptide of a-kafirins. Sequencing of kafirin PCR products revealed extensive edits in 25 of 26 events in one or multiple k1C family members. T1 and T2 seeds showed reduced a-kafirin levels, and selected T2 events showed significantly increased grain protein digestibility and lysine content. Thus, a single consensus single guide RNA carrying target sequence mismatches is sufficient for extensive editing of all k1C genes. The resulting quality improvements can be deployed rapidly for breeding and the generation of transgene-free, improved cultivars of sorghum, a major crop worldwide.

Sorghum (Sorghum bicolor) is a drought tolerant crop, which is being developed as a bioenergy feedstock. The monolignol biosynthesis pathway is a major focus for altering the abundance and composition of lignin. Caffeoyl coenzyme-A O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O-methyltransferase that methylates caffeoyl-CoA to generate feruloyl-CoA, an intermediate required for the biosynthesis of both G- and S-lignin. SbCCoAOMT was overexpressed to assess the impact of increasing the amount of this enzyme on biomass composition. SbCCoAOMT overexpression increased both soluble and cell wall-bound (esterified) ferulic and sinapic acids, however lignin concentration and its composition (S/G ratio) remained unaffected. This increased deposition of hydroxycinnamic acids in these lines led to an increase in total energy content of the stover. In stalk and leaf midribs, the increased histochemical staining and autofluorescence in the cell walls of the SbCCoAOMT overexpression lines also indicate increased phenolic deposition within cell walls, which is consistent with the chemical analyses of soluble and wall-bound hydroxycinnamic acids. The growth and development of overexpression lines were similar to wild-type plants. Likewise, RNA-seq and metabolite profiling showed that global gene expression and metabolite levels in overexpression lines were also relatively similar to wild-type plants. Our results demonstrate that SbCCoAOMT overexpression significantly altered cell wall composition through increases in cell wall associated hydroxycinnamic acids without altering lignin concentration or affecting plant growth and development.