Levi G. Lowder

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.

Genome editing promises giant leaps forward in advancing biotechnology, agriculture, and basic research. The process relies on the use of sequence specific nucleases (SSNs) to make DNA double stranded breaks at user defined genomic loci, which are subsequently repaired by two main DNA repair pathways: non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ can result in frameshift mutations that often create genetic knockouts. These knockout lines are useful for functional and reverse genetic studies but also have applications in agriculture. HDR has a variety of applications as it can be used for gene replacement, gene stacking, and for creating various fusion proteins. In recent years, transcription activator-like effector nucleases and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated protein 9 or CRISPR from Prevotella and Francisella 1 have emerged as the preferred SSNs for research purposes. Here, we review their applications in plant research, discuss current limitations, and predict future research directions in plant genome editing.