Study on a Novel Mini Genome Editing System Based on Csy4 and MCP
Jiahui Deng, Jianfeng Lei, Yi Zhao, Min Liu, Ziyao Hu, Yangzi You, Wukui Shao, Jianfei Liu, Xiaodong Liu
DOI: 10.13560/j.cnki.biotech.bull.1985.2023-0248
Genome editing technology refers to gene engineering techniques that utilize artificially constructed specific nucleases to modify the nucleic acid sequences of organisms through deletion, insertion, replacement, and other modifications. Currently, genome editing technologies mainly include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeat-associated protein) systems. Among them, second-generation genome editing technologies ZFNs and TALENs have achieved specific recognition of target sites and knockout of target genes, but their complex construction, high difficulty, long time consumption, high off-target rates, and low operability severely limit their application range.CRISPR/Cas technology has higher mutation efficiency, accuracy, and safety, and takes less time, but its large molecular weight of the targeted nuclease, off-target phenomena, and limitations imposed by PAM recognition sequences still require further improvement.
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In recent years, third-generation genome editing technologies represented by the CRISPR/Cas system have made significant progress in gene function research, plant molecular breeding, disease treatment, etc. However, the application fields of CRISPR/Cas series genome editing technologies still face issues such as large molecular weight of targeted nucleases, off-target phenomena, and limitations imposed by PAM recognition sequences. Researchers have been committed to finding solutions, improving gene editing efficiency, reducing off-target rates, and mitigating PAM site restrictions through a series of measures such as modifying Cas9 protein and using Cas9 orthologs. The MCP-FokI and Csy4-FokI fusion proteins constructed in this study may have advantages over Cas9 protein. This is mainly reflected in: small protein size, no PAM site restrictions, and low off-target efficiency. However, this system is a dual-target editing system, requiring the design of targeting sites on both sides of the target gene, thus presenting some complexity in design and vector construction. Additionally, the mini genome editing system may also be affected by the properties of the targeted proteins, such as the strength of RNA binding capability, RNA cutting activity, and the size of the targeted proteins, all of which may influence the editing capability of this system.
Virus-induced genome editing (VIGE) technology can modify plant viruses to express exogenous proteins and specific lengths of RNA in different plants. It can deliver genome editing components into plant cells through simple transformation methods, utilizing the systemic diffusion capability of viruses in plants, thus increasing the detection rate of gene editing. Cotton leaf crumple virus (CLCrV) is a bipartite virus transmitted by whiteflies, composed of two circular single-stranded DNA components, CLCrV-A and CLCrV-B. Therefore, this study attempts to construct the complete editing system onto the CLCrV virus vector, using the CLCrV virus vector to quickly verify the feasibility of the Csy4-FokI gene editing system in Arabidopsis leaves.
The MCP-FokI genome editing system constructed in this study did not detect gene editing occurrences through stable genetic transformation of Arabidopsis, and the reasons may include: whether the designed sgRNA and different intermediate spacing designs are effective, the method of protein fusion, and the strength of RNA binding capability. Among these, it is necessary to clarify the binding capability between MCP protein and MS2-guide-SL through detection methods for protein-RNA interactions. If the binding capability is weak, more MS2-SL can be tandemly added to enhance the binding capability of MCP protein with MS2-guide-SL, thereby improving editing efficiency.
For the Csy4-FokI editing system, the CLCrV virus vector was used to quickly verify the feasibility of the Csy4-FokI gene editing system in Arabidopsis leaves. The analysis of the results from Hi-TOM high-throughput detection revealed that the editing efficiency of the CLCrV-mediated Csy4-FokI editing system is low. Future research will focus on modifying Csy4 protein to eliminate its RNA cutting activity and only serve as an RNA binding protein to further validate the editing efficiency of this mini genome editing system. Future studies may also attempt to validate more genes and design different intermediate spacings, employing stable transformation methods such as floral dip to verify editing capability.
A new type of genome editing system without PAM site restrictions, Csy4-FokI mini genome editing system, has been preliminarily established. The CLCrV-mediated Csy4-FokI mini genome editing system can achieve targeted editing of the target gene, but the detected mutation types are limited to base substitutions, and the editing efficiency is very low.
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