Plant genome engineering with CRISPR-dCas12b system

Abstract

The environmental impacts of climate change are severe threats to agricultural systems and global food security. Biotechnological strategies comprise promising tools for plant molecular breeding, enabling the rapid and precise development of high-performance crops. In recent years, the CRISPR-Cas system has revolutionized plant biotechnology with its precision, feasibility, and low cost, fostering numerous functional genomics studies and the launch of genetically engineered products to the market. Still, these works are mostly dependent on loss-of-function applications, whereas gene overexpression approaches are still often limited to the barriers of conventional transgenic methodologies. Beyond genome editing, CRISPR activation (CRISPRa) can be conducted by deactivated Cas (dCas) enzymes coupled to activation domains, which recruit transcriptional activators onto promoter regions. This recent approach has been feasible in the simultaneous overexpression of multiple genes via CRISPR-dCas9 and -dCas12a systems. However, the dCas12b enzyme still shows low efficiency in plant transcriptional activation due to its components and mechanism of action. Considering that dCas12b has valuable molecular advantages, such as minimal off-target effects and smaller protein size, further efforts in optimizing the dCas12b-based activation system are warranted. In this regard, the present study aims to develop a highly-efficient CRISPR-dCas12b transcriptional system in rice (Oryza sativa) plants. First, the molecular components of the transcriptional activation system will be engineered in different configurations, cloned into an expression vector (via Golden Gate and Gateway® cloning), and then assessed in rice protoplasts. Once the system reaches high efficiency, rice stable lines will be genetically transformed and evaluated. For both approaches, gene expression profiles will be assessed by quantitative reverse-transcription PCR (RT-qPCR). Collectively, the outcomes of this study may be further extended to multiplex transcriptional activation and gene knockout applications, which can be easily translated to other economically important crops in the world. Due to the universality of the CRISPR technique, our study promises a versatile genome engineering tool with potential application not only in plants but in animals and fungi. (AU)