The recent progress of CRISPR/Cas genome editing technology and its application in crop improvement

Abstract

<p indent="0mm">CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) nucleases were originally identified in bacteria and archaea as their immune protection system to destroy phage and exogenous DNA. Relatively simple and powerful genome editing technologies have been developed based on the CRISPR/Cas system. In the last decade, CRISPR/Cas has been utilized as an effective genome editing tool in microorganisms, animals and plants. In this review, we will first discuss how the CRISPR/Cas system (e.g., CRISPR/Cas9) is used to edit plant genomes through various developed tools including small and large size DNA fragment deletions, homology-directed recombination, cytosine base editor, adenine base editor, dual base editor and prime editor. Details about these CRISPR/Cas plant genome editing tools including their working mechanisms, the construction of these tools using various functional elements, and several examples of applications will be provided. Indeed, the successful development of these tools allows researchers to modify plant genomes, functionally dissect plant genes, conduct molecular-design based breeding. Although the CRISPR/Cas T-DNA can be screened out either through back-crossing to wild type or self-crossing, the DNA-free CRISPR/Cas plant genome editing technologies bring obvious advantages in crop molecular breeding, especially for vegetative breeding crops or long juvenile stage plants. Therefore, secondly, we will describe how DNA-free CRISPR/Cas plant genome editing technology works. DNA-free CRISPR/Cas plant genome editing technology, which is especially useful in crop molecular breeding, can be achieved through the following approaches: Transiently expressed CRISPR/Cas DNA can be delivered into immature embryos using particle bombardment, or into explants using <italic>Agrobacterium tumefacien</italic> mediated method, or into protoplasts through polyethylene glycol mediation; <italic>in vitro</italic> transcripts of sgRNA and Cas nuclease can be imported into immature embryos using particle bombardment. BothsgRNA and Cas nuclease can be cloned into RNA virus vector before they are used to transform explants. Ribonucleoproteins<italic> </italic>complexes formed by <italic>in vitro </italic>transcribed<italic> </italic>sgRNA and Cas nuclease can be delivered into protoplasts, immature embryos through polyethylene glycol, lipofection or nanoparticle mediated methods, respectively. DNA-free edited plants can be identified from plants regenerated on medium without selective reagent. Thirdly, we will cover the recent successful applications of CRISPR/Cas as a plant genome-editing tool in crop improvements including crop yield and quality improvement, biotic and abiotic stresses resistance, de novo domestication, and crop directed improvement. These examples demonstrate that CRISPR/Cas plant genome editing technology is effective and valuable to crop molecular breeding. Lastly, we will discuss the future development of CRISPR/Cas technology in plant genome editing, the role of appropriate national management policies and favorable social environment in promoting this field. The editing efficiency of these tools can be further improved and the off-target rate reduced. Further enhancement of the CRISPR/Cas technology will depend on improved functional elements (e.g., CasX, SuperFi-Cas9, engineered APOBEC3A and TadA, etc.) and good vector design. Successful applications of CRISPR/Cas plant genome editing technologies in crop improvement will depend on appropriate regulatory policies and public acceptance of genome-edited crops. This paper aims to provide important insights into how CRISPR/Cas as a powerful plant genome-editing technology can be used in improving crop varieties, accelerating seed industry development, and fulfilling our national strategy of food storage in technology.