Nicholas J. Baltes

Cryptococcal cell morphology affects host cell interactions and pathogenicity.

Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 µm. Cell enlargement was observed in vivo, producing cells up to 100 µm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3 a Δ pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.

DNA Replicons for Plant Genome Engineering

The ability to precisely modify DNA in cells offers great opportunities for basic and applied research, yet it remains difficult to achieve for most plant species. This work explores the use of geminiviruses for genome engineering. Properties of geminiviruses, including extrachromosomal replication and pleiotropic activity of virus proteins, enabled highly efficient editing of the tobacco genome. Sequence-specific nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of plant genomes remains challenging due to ineffective methods for delivering reagents for genome engineering to plant cells. Here, we use geminivirus-based replicons for transient expression of sequence-specific nucleases (zinc-finger nucleases, transcription activator–like effector nucleases, and the clustered, regularly interspaced, short palindromic repeat/Cas system) and delivery of DNA repair templates. In tobacco (Nicotiana tabacum), replicons based on the bean yellow dwarf virus enhanced gene targeting frequencies one to two orders of magnitude over conventional Agrobacterium tumefaciens T-DNA. In addition to the nuclease-mediated DNA double-strand breaks, gene targeting was promoted by replication of the repair template and pleiotropic activity of the geminivirus replication initiator proteins. We demonstrate the feasibility of using geminivirus replicons to generate plants with a desired DNA sequence modification. By adopting a general plant transformation method, plantlets with a desired DNA change were regenerated in <6 weeks. These results, in addition to the large host range of geminiviruses, advocate the use of replicons for plant genome engineering.

Targeted Mutagenesis of Duplicated Genes in Soybean with Zinc-Finger Nucleases

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform—a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting DICER-LIKE (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome.

A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation

A CRISPR/Cas9 toolbox enables multiplex genome editing and transcriptional regulation of genes in plants. The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.

High-frequency, precise modification of the tomato genome

BackgroundThe use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA.ResultsHere, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences.ConclusionsHigh-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA.

Enabling plant synthetic biology through genome engineering

Synthetic biology seeks to create new biological systems, including user-designed plants and plant cells. These systems can be employed for a variety of purposes, ranging from producing compounds of industrial or therapeutic value, to reducing crop losses by altering cellular responses to pathogens or climate change. To realize the full potential of plant synthetic biology, techniques are required that provide control over the genetic code - enabling targeted modifications to DNA sequences within living plant cells. Such control is now within reach owing to recent advances in the use of sequence-specific nucleases to precisely engineer genomes. We discuss here the enormous potential provided by genome engineering for plant synthetic biology.

Improving cold storage and processing traits in potato through targeted gene knockout

Cold storage of potato tubers is commonly used to reduce sprouting and extend postharvest shelf life. However, cold temperature stimulates the accumulation of reducing sugars in potato tubers. Upon high-temperature processing, these reducing sugars react with free amino acids, resulting in brown, bitter-tasting products and elevated levels of acrylamide--a potential carcinogen. To minimize the accumulation of reducing sugars, RNA interference (RNAi) technology was used to silence the vacuolar invertase gene (VInv), which encodes a protein that breaks down sucrose to glucose and fructose. Because RNAi often results in incomplete gene silencing and requires the plant to be transgenic, here we used transcription activator-like effector nucleases (TALENs) to knockout VInv within the commercial potato variety, Ranger Russet. We isolated 18 plants containing mutations in at least one VInv allele, and five of these plants had mutations in all VInv alleles. Tubers from full VInv-knockout plants had undetectable levels of reducing sugars, and processed chips contained reduced levels of acrylamide and were lightly coloured. Furthermore, seven of the 18 modified plant lines appeared to contain no TALEN DNA insertions in the potato genome. These results provide a framework for using TALENs to quickly improve traits in commercially relevant autotetraploid potato lines.

Conferring resistance to geminiviruses with the CRISPR–Cas prokaryotic immune system

To reduce crop losses due to geminivirus infection, we targeted the bean yellow dwarf virus (BeYDV) genome for destruction with the CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins) system. Transient assays using BeYDV-based replicons revealed that CRISPR–Cas reagents introduced mutations within the viral genome and reduced virus copy number. Transgenic plants expressing CRISPR–Cas reagents and challenged with BeYDV had reduced virus load and symptoms, thereby demonstrating a novel strategy for engineering resistance to geminiviruses.

Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System

Targeted genome editing in plants will not only facilitate functional genomics studies but also help to discover, expand, and create novel traits of agricultural importance (Pennisi, 2010). The most widely used approach for editing plant genomes involves generating targeted double-strand DNA breaks (DSBs) and harnessing the two main DSB repair pathways: imprecise non-homologous end joining and precise homology-directed repair (Voytas, 2013). Enzymes that specifically bind the user-selected genomic sequences to create DSBs can be generated de novo as synthetic bimodular proteins containing a DNA-binding module, engineered to bind a user-defined sequence, along with a DNA-cleaving module, capable of making DSBs.

High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.