A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation

Abstract

The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated 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.

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

“…Furthermore, the development and customization of plant CRISPR/Cas9 toolboxes or platforms result in more powerful molecular tools for fundamental research and crop breeding (Lowder et al ., 2015; Ma et al ., 2015; Mao et al ., 2016; Tang et al ., 2016; Wang et al ., 2015; Xie et al ., 2015; Xing et al ., 2014). Rice is the most frequently used crop species for testing and applying CRISPR‐Cas9 tools.…”

Synthetic Botany

“…Notably, CRISPR-Cas9 genome editing has been successfully used to promote homologous recombination and to simultaneously target multiple loci in Arabidopsis and Nicotiana (Li et al 2014), as well as to induce targeted deletions in Nicotiana and rice (Lowder et al 2015). Such methods are enabling unprecedented efficiency of construct delivery, genome refactoring, and chassis engineering in plants.…”

“…Other useful circuits comprise the Cre-Lox recombinase system that can be used for induction of transcription (Hoff et al 2001) or transgene excision (Chakraborti et al 2008), and a catalytically inactive version of Cas9 fused to regulatory domains. The latter has been shown to perform effective transcriptional activation and repression of an endogenous gene in Nicotiana (Piatek et al 2015) and Arabidopsis (Lowder et al 2015). Posttranscriptional control over gene expression in plants has been enabled by a variety of RNA-based technologies.…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…SgRNAs are typically expressed from Pol III promoters (usually from the U6 or U3 genes coding for small nuclear RNAs as described previously) to prevent final polyadenylation, whereas the Cas9 protein is expressed from a Pol II promoter [83]. To synchronize the expression of these two types of elements and simplify the multiplexing of sgRNAs (that can require multiple Pol III promoters that are not always well-characterized in all species), they can be put under the control of a single Pol II promoter, which also provides greater flexibility for constitutive or inducible expression (Fig.…”

“…In this system, the transcript is cleaved using endogenous tRNA processing ribonucleases, RNases P and Z. Expression from Pol II promoters of this type of tRNA polycistronic sgRNAs has not yet been demonstrated in plants [83]. Another approach, still not tested in plants, could consist in replacing the tRNA motifs by sequences of 28 bp recognized by Csy4, a CRISPR type III ribonuclease [87].…”