Plant Cell:植物中的CRISPR筛选：方法，指南和未来展望

CRISPR-Cas系统通过促进广泛的靶向DNA扰动而彻底改变了基因组工程。这些系统已经产生了功能强大的新型筛选，可以在基因组规模上测试基因功能。尽管CRISPR筛查技术具有以空前的速度和规模进行基因定位和讯问的巨大潜力，但它们在植物中的应用仍处于起步阶段。

在这里，我们讨论了在植物中建立CRISPR筛选的一般概念，工具和工作流程，并分析了使用这种策略生成突变基因敲除集合或使DNA序列多样化的最新报告。

此外，鉴于当前的挑战和局限性，我们提供了有关如何在植物中设计CRISPR基因敲除筛选的见解，并探讨了多种设计方案。

最后，我们讨论了CRISPR筛选的独特多重功能，以研究高度重复的植物基因组中的冗余基因功能。组合突变体筛选具有常规产生更高阶突变体集合并促进基因网络表征的潜力。通过将这种方法与近二十年来产生的大量基因组图谱资源进行整合，CRISPR筛选的实施为以更深的分辨率分析植物基因组提供了新的机会，并将极大地促进植物功能和合成生物学。

CRISPR-Cas systems have revolutionized genome engineering by facilitating a wide range of targeted DNA perturbations. These systems have resulted in new powerful screens to test gene functions at the genomic scale. While there is tremendous potential for CRISPR screens to map and interrogate gene regulatory networks at unprecedented speed and scale, their implementation in plants remains in its infancy. Here we discuss the general concepts, tools and workflows for establishing CRISPR screens in plants and analyze the handful of recent reports using this strategy to generate mutant knockout collections or diversify DNA sequences. In addition, we provide insight on how to design CRISPR knockout screens in plants given the current challenges and limitations and examine multiple design options. Finally, we discuss the unique multiplexing capabilities of CRISPR screens to investigate redundant gene function in highly duplicated plant genomes. Combinatorial mutant screens have the potential to routinely generate higher-order mutant collections and facilitate the characterization of gene networks. By integrating this approach with the large resource of genomic profiles that were generated in the last two decades, the implementation of CRISPR screens offers new opportunities to analyze plant genomes at deeper resolution and will greatly advance plant functional and synthetic biology.

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