Discovery of deaminase functions by structure-based protein clustering.

The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected deaminase proteins to analyze and identified many previously unknown properties. We were surprised to find that most proteins in the DddA-like clade were not double-stranded DNA deaminases. We engineered the smallest single-strand-specific cytidine deaminase, enabling efficient cytosine base editor (CBE) to be packaged into a single adeno-associated virus (AAV). Importantly, we profiled a deaminase from this clade that edits robustly in soybean plants, which previously was inaccessible to CBEs. These discovered deaminases, based on AI-assisted structural predictions, greatly expand the utility of base editors for therapeutic and agricultural applications.

Progress in Soybean Genetic Transformation Over the Last Decade.

Soybean is one of the important food, feed, and biofuel crops in the world. Soybean genome modification by genetic transformation has been carried out for trait improvement for more than 4 decades. However, compared to other major crops such as rice, soybean is still recalcitrant to genetic transformation, and transgenic soybean production has been hampered by limitations such as low transformation efficiency and genotype specificity, and prolonged and tedious protocols. The primary goal in soybean transformation over the last decade is to achieve high efficiency and genotype flexibility. Soybean transformation has been improved by modifying tissue culture conditions such as selection of explant types, adjustment of culture medium components and choice of selection reagents, as well as better understanding the transformation mechanisms of specific approaches such as Agrobacterium infection. Transgenesis-based breeding of soybean varieties with new traits is now possible by development of improved protocols. In this review, we summarize the developments in soybean genetic transformation to date, especially focusing on the progress made using Agrobacterium-mediated methods and biolistic methods over the past decade. We also discuss current challenges and future directions.

Genetic Dissection of CRISPR-Cas9 Mediated Inheritance of Independently Targeted Alleles in Tobacco α-1,3-Fucosyltransferase 1 and ß-1,2-Xylosyltransferase 1 Loci.

We determined the specificity of mutations induced by the CRISPR-Cas9 gene-editing system in tobacco (Nicotiana benthamiana) alleles and subsequent genetic stability. For this, we prepared 248 mutant plants using an Agrobacterium-delivered CRISPR-Cas9 system targeting α-1,3-fucosyltransferase 1 (FucT1) and ß-1,2-xylosyltransferase1 (XylT1) genes, for which the mutation rates were 22.5% and 25%, respectively, with 20.5% for both loci. Individuals with wild-type (WT) alleles at the NbFucT1 locus in T0 were further segregated into chimeric progeny (37-54%) in the next generation, whereas homozygous T0 mutants tended to produce more (~70%) homozygotes than other bi-allelic and chimeric progenies in the T1 generation. Approximately 81.8% and 77.4% of the homozygous and bi-allelic mutations in T0 generation, respectively, were stably inherited in the next generation, and approximately 50% of the Cas9-free mutants were segregated in T2 generation. One homozygous mutant (Ta 161-1) with a +1 bp insertion in NbFucT1 and a -4 bp deletion in NbXylT1 was found to produce T2 progenies with the same alleles, indicating no activity of the integrated Cas9 irrespective of the insertion or deletion type. Our results provide empirical evidence regarding the genetic inheritance of alleles at CRISPR-targeted loci in tobacco transformants and indicate the potential factors contributing to further mutagenesis.

Efficient Genome Editing in Setaria italica Using CRISPR/Cas9 and Base Editors.

The genome editing toolbox based on CRISPR/Cas9 has brought revolutionary changes to agricultural and plant scientific research. With the development of stable genetic transformation protocols, a highly efficient genome editing system for foxtail millet (Setaria italica) is required. In the present study, we use the CRISPR/Cas9 single- and multi-gene knockout system to target the SiFMBP, SiDof4, SiBADH2, SiGBSS1, and SiIPK1 genes in the foxtail millet protoplasts to screen out highly efficient targeted sgRNAs. Then, we recovered homozygous mutant plants with most of the targeted genes through an Agrobacterium-mediated genetic transformation of foxtail millet. The mutagenesis frequency in the T0 generation was as high as 100%, and it was passed stably on to the next generation. After screening these targeted edited events, we did not detect off-target mutations at potential sites. Based on this system, we have achieved base editing successfully using two base editors (CBE and ABE) to target the SiALS and SiACC genes of foxtail millet. By utilizing CBE to target the SiALS gene, we created a homozygous herbicide-tolerant mutant plant. The current system could enhance the analysis of functional genomics and genetic improvement of foxtail millet.

Progresses, Challenges, and Prospects of Genome Editing in Soybean (Glycine max).

Soybean is grown worldwide for oil and protein source as food, feed and industrial raw material for biofuel. Steady increase in soybean production in the past century mainly attributes to genetic mediation including hybridization, mutagenesis and transgenesis. However, genetic resource limitation and intricate social issues in use of transgenic technology impede soybean improvement to meet rapid increases in global demand for soybean products. New approaches in genomics and development of site-specific nucleases (SSNs) based genome editing technologies have expanded soybean genetic variations in its germplasm and have potential to make precise modification of genes controlling the important agronomic traits in an elite background. ZFNs, TALENS and CRISPR/Cas9 have been adapted in soybean improvement for targeted deletions, additions, replacements and corrections in the genome. The availability of reference genome assembly and genomic resources increases feasibility in using current genome editing technologies and their new development. This review summarizes the status of genome editing in soybean improvement and future directions in this field.

Zinc finger nuclease-mediated precision genome editing of an endogenous gene in hexaploid bread wheat (Triticum aestivum) using a DNA repair template.

Sequence-specific nucleases have been used to engineer targeted genome modifications in various plants. While targeted gene knockouts resulting in loss of function have been reported with relatively high rates of success, targeted gene editing using an exogenously supplied DNA repair template and site-specific transgene integration has been more challenging. Here, we report the first application of zinc finger nuclease (ZFN)-mediated, nonhomologous end-joining (NHEJ)-directed editing of a native gene in allohexaploid bread wheat to introduce, via a supplied DNA repair template, a specific single amino acid change into the coding sequence of acetohydroxyacid synthase (AHAS) to confer resistance to imidazolinone herbicides. We recovered edited wheat plants having the targeted amino acid modification in one or more AHAS homoalleles via direct selection for resistance to imazamox, an AHAS-inhibiting imidazolinone herbicide. Using a cotransformation strategy based on chemical selection for an exogenous marker, we achieved a 1.2% recovery rate of edited plants having the desired amino acid change and a 2.9% recovery of plants with targeted mutations at the AHAS locus resulting in a loss-of-function gene knockout. The latter results demonstrate a broadly applicable approach to introduce targeted modifications into native genes for nonselectable traits. All ZFN-mediated changes were faithfully transmitted to the next generation.

Current and future editing reagent delivery systems for plant genome editing.

Many genome editing tools have been developed and new ones are anticipated; some have been extensively applied in plant genetics, biotechnology and breeding, especially the CRISPR/Cas9 system. These technologies have opened up a new era for crop improvement due to their precise editing of user-specified sequences related to agronomic traits. In this review, we will focus on an update of recent developments in the methodologies of editing reagent delivery, and consider the pros and cons of current delivery systems. Finally, we will reflect on possible future directions.

Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion.

Targeted base editing in plants without the need for a foreign DNA donor or double-stranded DNA cleavage would accelerate genome modification and breeding in a wide array of crops. We used a CRISPR-Cas9 nickase-cytidine deaminase fusion to achieve targeted conversion of cytosine to thymine from position 3 to 9 within the protospacer in both protoplasts and regenerated rice, wheat and maize plants at frequencies of up to 43.48%.

Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes.

Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

Reduced lignin content and altered lignin composition in the warm season forage grass Paspalum dilatatum by down-regulation of a Cinnamoyl CoA reductase gene.

C4 grasses are favoured as forage crops in warm, humid climates. The use of C4 grasses in pastures is expected to increase because the tropical belt is widening due to global climate change. While the forage quality of Paspalum dilatatum (dallisgrass) is higher than that of other C4 forage grass species, digestibility of warm-season grasses is, in general, poor compared with most temperate grasses. The presence of thick-walled parenchyma bundle-sheath cells around the vascular bundles found in the C4 forage grasses are associated with the deposition of lignin polymers in cell walls. High lignin content correlates negatively with digestibility, which is further reduced by a high ratio of syringyl (S) to guaiacyl (G) lignin subunits. Cinnamoyl-CoA reductase (CCR) catalyses the conversion of cinnamoyl CoA to cinnemaldehyde in the monolignol biosynthetic pathway and is considered to be the first step in the lignin-specific branch of the phenylpropanoid pathway. We have isolated three putative CCR1 cDNAs from P. dilatatum and demonstrated that their spatio-temporal expression pattern correlates with the developmental profile of lignin deposition. Further, transgenic P. dilatatum plants were produced in which a sense-suppression gene cassette, delivered free of vector backbone and integrated separately to the selectable marker, reduced CCR1 transcript levels. This resulted in the reduction of lignin, largely attributable to a decrease in G lignin.

Functional analyses of caffeic acid O-Methyltransferase and Cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne).

Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.

A high throughput Agrobacterium tumefaciens-mediated transformation method for functional genomics of perennial ryegrass (Lolium perenne L.).

A robust and high throughput Agrobacterium genetic transformation procedure has been developed for perennial ryegrass (Lolium perenne L.). Embryogenic callus lines were selected and maintained as plants in vitro. Embryogenic calli derived from meristematic regions of the vegetative tillers were co-cultivated with Agrobacterium tumefaciens strain EHA101 carrying the plasmid pCAMBIA 1305.1 in the presence of acetosyringone for 3-4 days. The calli were grown under 94.8 and 151.6 microM hygromycin selection, respectively for two cycles of 2-weeks each, followed by transfer to regeneration medium with 47.4 microM hygromycin. Regenerated plants were rooted and successfully transferred to soil. The transgenic nature of the regenerated plants was confirmed by DNA gel blot analysis and gene expression demonstrated by GUS histochemical assay and/or reverse transcription PCR. After development of the transformation procedure, we used Agrobacterium strain EHA101 carrying a modified binary plasmid pMH bearing genes of interest. In the past 2 years, we have produced more than 1,000 plants with constructs encoding different genes of interest from perennial ryegrass.

Transformation of Actinidia eriantha: a potential species for functional genomics studies in Actinidia.

Protocols were developed for regeneration and Agrobacterium-mediated transformation of Actinidia eriantha Benth. A. eriantha has a number of features that make it a useful tool for functional genomics in Actinidia: the vines are relatively small and non-vigorous in nature, flowers form all over the vine including on lower axillary branches and the species flowers prolifically in greenhouse conditions. Flowering and fruiting of transgenic A. eriantha plants was obtained within 2 years of transformation in a containment greenhouse. GUS (beta-glucuronidase) activity indicating stable expression of the uidA gene was observed in leaf, stem, root, petal and fruit tissues. Molecular evidence for incorporation of transgenes into the A. eriantha genome was obtained by PCR and DNA gel blot analysis. Inheritance of transgenic phenotypes was demonstrated in seedling progeny. Functional genomic studies in kiwifruit have been initiated using transgenic A. eriantha plants.