Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous doublestranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)— in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ~1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonasacetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures

Dicamba Resistance: Enlarging and Preserving Biotechnology-Based Weed Management Strategies

The advent of biotechnology-derived, herbicide-resistant crops has revolutionized farming practices in many countries. Facile, highly effective, environmentally sound, and profitable weed control methods have been rapidly adopted by crop producers who value the benefits associated with biotechnology-derived weed management traits. But a rapid rise in the populations of several troublesome weeds that are tolerant or resistant to herbicides currently used in conjunction with herbicide-resistant crops may signify that the useful lifetime of these economically important weed management traits will be cut short. We describe the development of soybean and other broadleaf plant species resistant to dicamba, a widely used, inexpensive, and environmentally safe herbicide. The dicamba resistance technology will augment current herbicide resistance technologies and extend their effective lifetime. Attributes of both nuclear- and chloroplast- encoded dicamba resistance genes that affect the potency and expected durability of the herbicide resistance trait are examined

Dicamba Resistance: Enlarging and Preserving Biotechnology-Based Weed Management Strategies

The advent of biotechnology-derived, herbicide-resistant crops has revolutionized farming practices in many countries. Facile, highly effective, environmentally sound, and profitable weed control methods have been rapidly adopted by crop producers who value the benefits associated with biotechnology-derived weed management traits. But a rapid rise in the populations of several troublesome weeds that are tolerant or resistant to herbicides currently used in conjunction with herbicide-resistant crops may signify that the useful lifetime of these economically important weed management traits will be cut short. We describe the development of soybean and other broadleaf plant species resistant to dicamba, a widely used, inexpensive, and environmentally safe herbicide. The dicamba resistance technology will augment current herbicide resistance technologies and extend their effective lifetime. Attributes of both nuclear- and chloroplast- encoded dicamba resistance genes that affect the potency and expected durability of the herbicide resistance trait are examined

A rapid live-cell ELISA for characterizing antibodies against cell surface antigens of Chlamydomonas reinhardtii and its use in isolating algae from natural environments with related cell wall components

Background: Cell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors. In the case of eukaryotic algae, relatively little is known of the composition, structure or mechanisms of assembly of cell walls in individual species or between species and how these differences enable algae to inhabit a great diversity of environments. In this paper we describe the use of camelid antibody fragments (VHHs) and a streamlined ELISA assay as powerful new tools for obtaining mono-specific reagents for detecting individual algal cell wall components and for isolating algae that share a particular cell surface component. Results: To develop new microalgal bioprospecting tools to aid in the search of environmental samples for algae that share similar cell wall and cell surface components, we have produced single-chain camelid antibodies raised against cell surface components of the single-cell alga, Chlamydomonas reinhardtii. We have cloned the variable-region domains (V[subscript H]Hs) from the camelid heavy-chain-only antibodies and overproduced tagged versions of these monoclonal-like antibodies in E. coli. Using these V[subscript H]Hs, we have developed an accurate, facile, low cost ELISA that uses live cells as a source of antigens in their native conformation and that requires less than 90 minutes to perform. This ELISA technique was demonstrated to be as accurate as standard ELISAs that employ proteins from cell lysates and that generally require >24 hours to complete. Among the cloned V[subscript H]Hs, V[subscript H]H B11, exhibited the highest affinity (EC[subscript 50] < 1 nM) for the C. reinhardtii cell surface. The live-cell ELISA procedure was employed to detect algae sharing cell surface components with C. reinhardtii in water samples from natural environments. In addition, mCherry-tagged V[subscript H]H B11 was used along with fluorescence activated cell sorting (FACS) to select individual axenic isolates of presumed wild relatives of C. reinhardtii and other Chlorphyceae from the same environmental samples. Conclusions: Camelid antibody V[subscript H]H domains provide a highly specific tool for detection of individual cell wall components of algae and for allowing the selection of algae that share a particular cell surface molecule from diverse ecosystems

SWIM: A Semi-Analytical Ocean Color Inversion Algorithm for Optically Shallow Waters

Ocean color remote sensing provides synoptic-scale, near-daily observations of marine inherent optical properties (IOPs). Whilst contemporary ocean color algorithms are known to perform well in deep oceanic waters, they have difficulty operating in optically clear, shallow marine environments where light reflected from the seafloor contributes to the water-leaving radiance. The effect of benthic reflectance in optically shallow waters is known to adversely affect algorithms developed for optically deep waters [1, 2]. Whilst adapted versions of optically deep ocean color algorithms have been applied to optically shallow regions with reasonable success [3], there is presently no approach that directly corrects for bottom reflectance using existing knowledge of bathymetry and benthic albedo.To address the issue of optically shallow waters, we have developed a semi-analytical ocean color inversion algorithm: the Shallow Water Inversion Model (SWIM). SWIM uses existing bathymetry and a derived benthic albedo map to correct for bottom reflectance using the semi-analytical model of Lee et al [4]. The algorithm was incorporated into the NASA Ocean Biology Processing Groups L2GEN program and tested in optically shallow waters of the Great Barrier Reef, Australia. In-lieu of readily available in situ matchup data, we present a comparison between SWIM and two contemporary ocean color algorithms, the Generalized Inherent Optical Property Algorithm (GIOP) and the Quasi-Analytical Algorithm (QAA)

Enhancing soybean photosynthetic CO2 assimilation using a cyanobacterial membrane protein, ictB

Abstract Soybean C3 photosynthesis can suffer a severe loss in efficiency due to photorespiration and the lack of a carbon concentrating mechanism (CCM) such as those present in other plant species or cyanobacteria. Transgenic soybean (Glycine max cv. Thorne) plants constitutively expressing cyanobacterial ictB (inorganic carbon transporter B) gene were generated using Agrobacterium-mediated transformation. Although more recent data suggest that ictB does not actively transport HCO3-/CO2, there is nevertheless mounting evidence that transformation with this gene can increase higher plant photosynthesis. The hypothesis that expression of the ictB gene would improve photosynthesis, biomass production and seed yield in soybean was tested, in two independent replicated greenhouse and field trials. Results showed significant increases in photosynthetic CO2 uptake (Anet) and dry mass in transgenic relative to wild type (WT) control plants in both the greenhouse and field trials. Transgenic plants also showed increased photosynthetic rates and biomass production during a drought mimic study. The findings presented herein demonstrate that ictB, as a single-gene, contributes to enhancement in various yield parameters in a major commodity crop and point to the significant role that biotechnological approaches to increasing photosynthetic efficiency can play in helping to meet increased global demands for food

SWIM: A Semi-Analytical Ocean Color Inversion Algorithm for Optically Shallow Waters

In clear shallow waters, light that is transmitted downward through the water column can reflect off the sea floor and thereby influence the water-leaving radiance signal. This effect can confound contemporary ocean color algorithms designed for deep waters where the seafloor has little or no effect on the water-leaving radiance. Thus, inappropriate use of deep water ocean color algorithms in optically shallow regions can lead to inaccurate retrievals of inherent optical properties (IOPs) and therefore have a detrimental impact on IOP-based estimates of marine parameters, including chlorophyll-a and the diffuse attenuation coefficient. In order to improve IOP retrievals in optically shallow regions, a semi-analytical inversion algorithm, the Shallow Water Inversion Model (SWIM), has been developed. Unlike established ocean color algorithms, SWIM considers both the water column depth and the benthic albedo. A radiative transfer study was conducted that demonstrated how SWIM and two contemporary ocean color algorithms, the Generalized Inherent Optical Properties algorithm (GIOP) and Quasi-Analytical Algorithm (QAA), performed in optically deep and shallow scenarios. The results showed that SWIM performed well, whilst both GIOP and QAA showed distinct positive bias in IOP retrievals in optically shallow waters. The SWIM algorithm was also applied to a test region: the Great Barrier Reef, Australia. Using a single test scene and time series data collected by NASA's MODIS-Aqua sensor (2002-2013), a comparison of IOPs retrieved by SWIM, GIOP and QAA was conducted

Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a ‘modular assembly’ method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry

TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain

DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination