CRISPR-Based Genome Editing Tools: An Accelerator in Crop Breeding for a Changing Future

Plant breeding has significantly advanced since the 1980s, progressing from transgenic methods to molecular marker systems and genomics approaches. While these methods have been successful in improving crop yields and quality, they are limited by the available genetic variation within existing crop gene pools. Gene editing techniques, particularly CRISPR-Cas systems, offer a powerful solution to overcome this limitation. These systems enable targeted mutagenesis, allowing for the rapid and precise generation of new crop varieties with improved traits. The increasing availability of crop genome data further enhances the applicability and efficiency of gene editing in crop improvement, enabling genetic modifications targeted at the gene level to improve crop quality and yield.

The paper reviews various gene-editing methods, beginning with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). ZFNs utilize zinc-binding domains to grasp DNA, fused with a DNA-cutting function. TALENs, derived from Xanthomonas effector proteins, also involve DNA-protein interactions linked to an endonuclease. The review then extensively covers CRISPR-Cas systems, highlighting their three stages: adaptation (spacer acquisition), pre-crRNA expression and processing, and interference (target DNA/RNA cleavage). The two main classes of CRISPR-Cas systems (Class 1 and Class 2) and their subtypes are classified based on the effector module composition (multi-subunit complex vs. single protein). The review also delves into the different DNA repair pathways involved after double-strand breaks (DSBs) are introduced: non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology-directed repair (HDR).

The paper employs a review methodology, systematically analyzing and synthesizing existing literature on CRISPR-Cas systems and their applications in plant genome editing. It begins with an overview of the historical development of plant breeding techniques and the limitations of traditional methods. The core of the review focuses on the detailed explanation of CRISPR-Cas systems, including their classification (Class 1 and Class 2), types (I, II, III, IV, V, VI), and subtypes. For each type and subtype, the paper describes the key components (Cas proteins, crRNAs, tracrRNAs), their mechanisms of action, and their applications in plant genome editing. This includes detailed explanations of Cas protein structures, domains, and functions. The authors compare and contrast different CRISPR-Cas systems, highlighting their respective advantages and limitations in terms of editing efficiency, target specificity, and ease of use. The review also includes discussions of various applications, such as gene knockout, knock-in, base editing, prime editing, CRISPRi, and CRISPRa. The paper systematically explains each technique's mechanism and its success rate in various plant species. Additionally, the paper describes the potential and current uses of CRISPR for tasks such as de novo domestication, gene stacking in polyploid crops, CRISPR screening, and gene drives.

The review highlights the diverse applications of CRISPR-Cas systems in plant genome editing. It details the successful use of CRISPR-Cas9 for generating completely sterile mutants in hexaploid wheat, demonstrating the potential for editing meiotic recombination. RNA-targeting SpCas9 (RCas9) and the use of PAMmers for precise RNA manipulation are also discussed. The review explains different base editing strategies and their use in modifying genes with high GC content, highlighting CRISPRi/CRISPRa systems for regulating gene expression. The use of Cas12a (Cpf1) for gene deletion, insertion, base editing, and locus tagging in various plants is discussed, along with a comparison of its efficiency with Cas9. The unique RNA-targeting capabilities of Cas13 are detailed, with examples of its use in targeted RNA knockdown, RNA virus defense, and epitranscriptome modification. The authors explore the potential of LwaCas13a and LshCas13a in rice and the exceptional efficiency of CasRx in tobacco. Class 1 type I CRISPR-Cas systems and their application in transcriptional control in maize are explored, alongside the hybrid type I-D system (TiD) and its ability to edit inaccessible genomic regions and create both small indels and long deletions. Type V-B CRISPR-Cas12b/C2c1 and its potential for efficient gene editing are described, including the high-mutation specificity of AaCas12b in rice and its function at high temperatures. The review concludes with a discussion of the hypercompact CRISPR-CasΦ system and its broad PAM recognition. The use of different CRISPR systems in de novo domestication, gene stacking in polyploids, CRISPR screening, and gene drives are discussed. Finally, the review examines the use of CRISPR/Cas9 in addressing food security issues, such as the generation of hybrid seeds and the improvement of crop nutrient uptake.

The findings of this review underscore the transformative potential of CRISPR-Cas systems for accelerating crop breeding and enhancing food security. The diverse range of CRISPR-Cas systems, each with its unique characteristics and applications, provides a powerful toolkit for addressing various challenges in plant breeding. The successful application of these systems in de novo domestication, gene stacking, and other advanced breeding approaches highlights their ability to overcome limitations of traditional methods. However, the review also acknowledges the challenges associated with CRISPR technology, including off-target effects, PAM sequence dependence, and the complexities of patent licensing. Addressing these challenges will be crucial for maximizing the benefits of CRISPR-Cas systems in plant breeding and ensuring equitable access to this technology.

CRISPR-Cas systems represent a potent tool for synthetic plant biology, particularly for creating synthetic plant genomes. The paper concludes that CRISPR systems are rapidly evolving and improving, with advancements continually addressing limitations such as target recognition and off-target effects. The authors believe that CRISPR-Cas systems will continue to become increasingly effective and flexible in plant genome editing, ultimately contributing to global food security in the face of a changing climate.

While the review provides a comprehensive overview of CRISPR-Cas systems and their applications, it is important to note that the field is rapidly evolving. Some systems, particularly Class 1 and Type IV systems, are less well-characterized than Class 2 systems. Furthermore, the practical applications of some CRISPR systems in plant breeding might still require further optimization and development before becoming widely adopted. The discussion of patent licensing and commercialization highlights a limitation to the technology's widespread adoption due to complex intellectual property rights.