Rapid and efficient in planta genome editing in sorghum using foxtail mosaic virus-mediated sgRNA delivery.

The requirement of in vitro tissue culture for the delivery of gene editing reagents limits the application of gene editing to commercially relevant varieties of many crop species. To overcome this bottleneck, plant RNA viruses have been deployed as versatile tools for in planta delivery of recombinant RNA. Viral delivery of single-guide RNAs (sgRNAs) to transgenic plants that stably express CRISPR-associated (Cas) endonuclease has been successfully used for targeted mutagenesis in several dicotyledonous and few monocotyledonous plants.

Greater aperture counteracts effects of reduced stomatal density on water use efficiency: a case study on sugarcane and meta-analysis.

Stomata regulate CO2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (gsw).

The extent of multiallelic, co-editing of LIGULELESS1 in highly polyploid sugarcane tunes leaf inclination angle and enables selection of the ideotype for biomass yield.

Sugarcane (Saccharum spp. hybrid) is a prime feedstock for commercial production of biofuel and table sugar. Optimizing canopy architecture for improved light capture has great potential for elevating biomass yield.

Advances in genomics and genome editing for improving strawberry ().

The cultivated strawberry, , is a recently domesticated fruit species of economic interest worldwide. As such, there is significant interest in continuous varietal improvement. Genomics-assisted improvement, including the use of DNA markers and genomic selection have facilitated significant improvements of numerous key traits during strawberry breeding.

Intron-mediated enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 expression in energycane promotes a step change for lipid accumulation in vegetative tissues.

Background: Metabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG.

dCas9-3xSRDX-mediated transcriptional repression in sugarcane.

Targeting dCas9 fused with the 3xSRDX effector to the 5'UTR leads to strong repression of magnesium chelatase in highly polyploid sugarcane.

Multi-allelic gene editing in an apomictic, tetraploid turf and forage grass ( Flüggé) using CRISPR/Cas9.

Polyploidy is common among grasses () and poses challenges for conventional breeding. Genome editing technology circumvents crossing and selfing, enabling targeted modifications to multiple gene copies in a single generation while maintaining the heterozygous context of many polyploid genomes. Bahiagrass ( Flüggé; 2=4=40) is an apomictic, tetraploid C4 species that is widely grown in the southeastern United States as forage in beef cattle production and utility turf.

Towards oilcane: preliminary field evaluation of metabolically engineered sugarcane with hyper-accumulation of triacylglycerol in vegetative tissues.

Unlabelled: We recently generated oilcane, a metabolically engineered sugarcane with hyper-accumulation of energy dense triacylglycerol in vegetative tissues. Refinement of this strategy in high biomass crops like sugarcane may result in elevated lipid yields that exceed traditional oilseed crops for biodiesel production. This is the first report of agronomic performance, stable co-expression of lipogenic factors, and TAG accumulation in transgenic sugarcane under field conditions.

Microbiome differences in sugarcane and metabolically engineered oilcane accessions and their implications for bioenergy production.

Oilcane is a metabolically engineered sugarcane (Saccharum spp. hybrid) that hyper-accumulates lipids in its vegetable biomass to provide an advanced feedstock for biodiesel production. The potential impact of hyper-accumulation of lipids in vegetable biomass on microbiomes and the consequences of altered microbiomes on plant growth and lipid accumulation have not been explored so far.

Targeted mutagenesis with sequence-specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects.

Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids.

Engineering triacylglycerol accumulation in duckweed (Lemna japonica).

Duckweeds are amongst the fastest growing of higher plants, making them attractive high-biomass targets for biofuel feedstock production. Their fronds have high rates of fatty acid synthesis to meet the demand for new membranes, but triacylglycerols (TAG) only accumulate to very low levels. Here we report on the engineering of Lemna japonica for the synthesis and accumulation of TAG in its fronds.

Metabolic engineering of energycane to hyperaccumulate lipids in vegetative biomass.

Background: The metabolic engineering of high-biomass crops for lipid production in their vegetative biomass has recently been proposed as a strategy to elevate energy density and lipid yields for biodiesel production. Energycane and sugarcane are highly polyploid, interspecific hybrids between Saccharum officinarum and Saccharum spontaneum that differ in the amount of ancestral contribution to their genomes. This results in greater biomass yield and persistence in energycane, which makes it the preferred target crop for biofuel production.

CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance.

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2 = 100 - 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9.

Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane.

Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane ( = 10-13), remains challenging due to the high level of genetic redundancy in these plants. Here, we report the efficient multiallelic editing of magnesium chelatase subunit I () in sugarcane.

CRISPR/Cas9 mediated targeted mutagenesis of LIGULELESS-1 in sorghum provides a rapidly scorable phenotype by altering leaf inclination angle.

Sorghum (Sorghum bicolor L. Moench) is one of the world's most cultivated cereal crops. Biotechnology approaches have great potential to complement traditional crop improvement.

Error-free recombination in sugarcane mediated by only 30 nucleotides of homology and CRISPR/Cas9 induced DNA breaks or Cre-recombinase.

Precision genome editing by homology directed repair has tremendous potential for crop improvement. This study describes in planta homologous recombination mediated by CRISPR/Cas9 induced DNA double strand break in proximity to a single short (∼30 nt) homology arm. The efficiency of CRISPR/Cas9-mediated recombination between two loxP sites was compared with Cre (Cyclization recombination enzyme) and codon-optimized Cre-mediated site-specific recombination in sugarcane.

Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane.

A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination.

Surveying the genome and constructing a high-density genetic map of napiergrass (Cenchrus purpureus Schumach).

Napiergrass (Cenchrus purpureus Schumach) is a tropical forage grass and a promising lignocellulosic biofuel feedstock due to its high biomass yield, persistence, and nutritive value. However, its utilization for breeding has lagged behind other crops due to limited genetic and genomic resources. In this study, next-generation sequencing was first used to survey the genome of napiergrass.

Largely enhanced bioethanol production through the combined use of lignin-modified sugarcane and xylose fermenting yeast strain.

The recalcitrant structure of lignocellulosic biomass is a major barrier in efficient biomass-to-ethanol bioconversion processes. The combination of feedstock engineering via modification in the lignin synthesis pathway of sugarcane and co-fermentation of xylose and glucose with a recombinant xylose utilizing yeast strain produced 148% more ethanol compared to that of the wild type biomass and control strain. The lignin reduced biomass led to a substantially increased release of fermentable sugars (glucose and xylose).

TALEN-mediated targeted mutagenesis of more than 100 COMT copies/alleles in highly polyploid sugarcane improves saccharification efficiency without compromising biomass yield.

Sugarcane is the world's most efficient feedstock for commercial production of bioethanol due to its superior biomass production and accumulation of sucrose in stems. Integrating first- and second-generation ethanol conversion processes will enhance the biofuel yield per unit area by utilizing both sucrose and cell wall-bound sugars for fermentation. RNAi suppression of the lignin biosynthetic gene caffeic acid O-methyltransferase (COMT) has been demonstrated to improve bioethanol production from lignocellulosic biomass.

In planta production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane.

Sugarcane (Saccharum sp. hybrids) is one of the most efficient and sustainable feedstocks for commercial production of fuel ethanol. Recent efforts focus on the integration of first and second generation bioethanol conversion technologies for sugarcane to increase biofuel yields.

Precision breeding for RNAi suppression of a major 4-coumarate:coenzyme A ligase gene improves cell wall saccharification from field grown sugarcane.

Sugarcane (Saccharum spp. hybrids) is a major feedstock for commercial bioethanol production. The recent integration of conversion technologies that utilize lignocellulosic sugarcane residues as well as sucrose from stem internodes has elevated bioethanol yields.

Advancing Crop Transformation in the Era of Genome Editing.

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process.

TALEN mediated targeted mutagenesis of the caffeic acid O-methyltransferase in highly polyploid sugarcane improves cell wall composition for production of bioethanol.

Sugarcane (Saccharum spp. hybrids) is a prime crop for commercial biofuel production. Advanced conversion technology utilizes both, sucrose accumulating in sugarcane stems as well as cell wall bound sugars for commercial ethanol production.

Metabolic engineering of sugarcane to accumulate energy-dense triacylglycerols in vegetative biomass.

Elevating the lipid content in vegetative tissues has emerged as a new strategy for increasing energy density and biofuel yield of crops. Storage lipids in contrast to structural and signaling lipids are mainly composed of glycerol esters of fatty acids, also known as triacylglycerol (TAG). TAGs are one of the most energy-rich and abundant forms of reduced carbon available in nature.