CRISPR/LbCas12a-Mediated Genome Editing in Soybean.

Currently methods for generating soybean edited lines are time-consuming, inefficient, and limited to certain genotypes. Here we describe a fast and highly efficient genome editing method based on CRISPR-Cas12a nuclease system in soybean. The method uses Agrobacterium-mediated transformation to deliver editing constructs and uses aadA or ALS genes as selectable marker. It only takes about 45 days to obtain greenhouse-ready edited plants at higher than 30% transformation efficiency and 50% editing rate. The method is applicable to other selectable markers including EPSPS and has low transgene chimera rate. The method is also genotype-flexible and has been applied to genome editing of several elite soybean varieties.

Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize.

Recent advances in the development of CRISPR-Cas genome editing technologies have made it possible to perform targeted mutagenesis and precise gene replacement in crop plants. CRISPR-Cas9 and CRISPR-Cas12a are two main types of widely used genome editing systems. However, when CRISPR-Cas12a editing machinery is expressed from a transgene, some chromosomal targets encountered low editing frequency in important crops like maize and soybean. Here, we report efficient methods to directly generate genome edited lines by delivering Cas12a-gRNA ribonucleoprotein complex (RNP) to immature maize embryos through particle bombardment in an elite maize variety. Genome edited lines were obtained at ~7% frequency without any selection during regeneration via biolistic delivery of Cas12a RNP into immature embryos. Strikingly, the gene editing rate was increased to 60% on average and up to 100% in some experiments when the Cas12a RNP was co-delivered with a PMI selectable marker gene cassette and the induced callus cultures were selected with mannose. We also show that use of higher activity Cas12a mutants resulted in improved editing efficiency in more recalcitrant target sequence. The advances described here provide useful tools for genetic improvement of maize.

Morphogenic Regulators and Their Application in Improving Plant Transformation.

Generation of plant lines with transgene or edited gene variants is the desired outcome of transformation technology. Conventional DNA-based plant transformation methods are the most commonly used technology but these approaches are limited to a small number of plant species with efficient transformation systems. The ideal transformation technologies are those that allow biotechnology applications across wide genetic background, especially within elite germplasm of major crop species. This chapter will briefly review key regulatory genes involved in plant morphogenesis with a focus on in vitro somatic embryogenesis and their application in improving plant transformation.

Repurposing Macromolecule Delivery Tools for Plant Genetic Modification in the Era of Precision Genome Engineering.

Efficient delivery of macromolecules into plant cells and tissues is important for both basic research and biotechnology product applications. In transgenic research, the goal is to deliver DNA molecules into regenerable cells and stably integrate them into the genome. Over the past 40 years, many macromolecule delivery methods have been studied. To generate transgenic plants, particle bombardment and Agrobacterium-mediated transformation are the methods of choice for DNA delivery. The rapid advance of genome editing technologies has generated new requirements on large biomolecule delivery and at the same time reinvigorated the development of new transformation technologies. Many of the gene delivery options that have been studied before are now being repurposed for delivering genome editing machinery for various applications. This article reviews the major progress in the development of tools for large biomolecule delivery into plant cells in the new era of precision genome engineering.

Advances in Agrobacterium-mediated Maize Transformation.

One of the major limitations of maize transformation is the isolation of a large number of immature embryos using the time-consuming manual extraction method. In this article, we describe a novel bulk embryo extraction method for fast isolation of a large number of embryos suitable for both biolistic- and Agrobacterium-mediated transformation. Optimal gene delivery and tissue culture conditions are also described for achieving high efficiency in Agrobacterium-mediated maize transformation using phosphomannose isomerase (PMI) as a selectable marker.

Maize transformation technology development for commercial event generation.

Maize is an important food and feed crop in many countries. It is also one of the most important target crops for the application of biotechnology. Currently, there are more biotech traits available on the market in maize than in any other crop. Generation of transgenic events is a crucial step in the development of biotech traits. For commercial applications, a high throughput transformation system producing a large number of high quality events in an elite genetic background is highly desirable. There has been tremendous progress in Agrobacterium-mediated maize transformation since the publication of the Ishida et al. (1996) paper and the technology has been widely adopted for transgenic event production by many labs around the world. We will review general efforts in establishing efficient maize transformation technologies useful for transgenic event production in trait research and development. The review will also discuss transformation systems used for generating commercial maize trait events currently on the market. As the number of traits is increasing steadily and two or more modes of action are used to control key pests, new tools are needed to efficiently transform vectors containing multiple trait genes. We will review general guidelines for assembling binary vectors for commercial transformation. Approaches to increase transformation efficiency and gene expression of large gene stack vectors will be discussed. Finally, recent studies of targeted genome modification and transgene insertion using different site-directed nuclease technologies will be reviewed.