上海交大陆钰明团队发表综述文章：植物大规模基因组编辑：方法、应用和未来展望

2023年1月9日，Current Opinion in Biotechnology （IF=9.74）在线发表了上海交通大学陆钰明团队的题为Large-scale genome editing in plants: approaches, applications, and future perspectives的综述文章。

该综述总结了利用CRISPR技术对植物进行全基因组的筛选的工作流程，包括选择合适的编辑工具、全基因组gRNA设计、池文库构建、大规模转化和高通量基因分型；还讨论了在筛选候选基因、优化时空表达、蛋白质活性进化以及建立全基因组基因敲除突变体文库方面的应用。

Figure 1. Workflow for CRISPR screens in plants. First, a proper genome editor is selected. These SpCas9-based editors can be used to achieve gene knockout (CRISPRko), sequence knock-in, and nucleotide(s) editing (base editors and prime editors), and can also be used to activate (CRISPRa) or repress (CRISPRi) gene expression at the transcriptional level (a). The size of a CRISPR screen depends on the number of target genes, which can be effectively reduced by appropriate bioinformatics analysis. gRNA can be obtained by bulk design with various programs, resulting in a gRNA dataset (b). gRNA oligos can be synthesized on a chip and then cloned into a proper construct to obtain an arrayed or pooled library (c), which could be used for massive transformations and to then generate a large number of plantlets (d). The gRNAs and the causal mutations in mutants can be genotyped in a high-throughput manner (e) before or after phenotype screening. The edited seeds can be managed to create a mutant library (f).

同时在考虑了目前CRISPR技术的全基因组筛选的挑战和限制之后，作者设想了一种以病毒为媒介的策略来改进CRISPR筛选。

Figure 2. A future view for virus-mediated CRISPR screens in plant. Pooled gRNAs are constructed into a viral vector that is used to bulk-infect growing plants with a proper MOI. Harvested seeds may be sorted using an automated sorting machine to collect edited seeds for subsequent screens and analysis.

带有gRNA的混合病毒可以通过适当的MOI（即感染复数，指的是感染时病毒与细胞数量的比值）来大量感染生长中的植物，以确保“一个细胞一个病毒粒子”，就像在哺乳动物中所实现的那样。一旦由特定gRNA编辑的细胞分化为生殖细胞，基因编辑后就可以传递给种子，然后就可以由自动分选机批量选择种子。这种形式的CRISPR筛选很容易升级。

随着CRISPR筛选在植物中的快速发展和广泛采用，必将推动植物种质资源的大幅增加，这将极大地促进植物的基础研究和作物遗传改良。

原文链接：

https://doi.org/10.1016/j.copbio.2022.102875.

本文转载自eplants

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