Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Key Laboratory of Molecular Biophysics of the Ministry of Education, Wuhan 430074, China.
Genome Res. 2023 May;33(5):798-809. doi: 10.1101/gr.277650.123. Epub 2023 Jun 8.
The recently constructed mutant libraries of diploid crops by the CRISPR-Cas9 system have provided abundant resources for functional genomics and crop breeding. However, because of the genome complexity, it is a big challenge to accomplish large-scale targeted mutagenesis in polyploid plants. Here, we demonstrate the feasibility of using a pooled CRISPR library to achieve genome-scale targeted editing in an allotetraploid crop of A total of 18,414 sgRNAs were designed to target 10,480 genes of interest, and afterward, 1104 regenerated transgenic plants harboring 1088 sgRNAs were obtained. Editing interrogation results revealed that 93 of the 178 genes were identified as mutated, thus representing an editing efficiency of 52.2%. Furthermore, we have discovered that Cas9-mediated DNA cleavages tend to occur at all the target sites guided by the same individual sgRNA, a novel finding in polyploid plants. Finally, we show the strong capability of reverse genetic screening for various traits with the postgenotyped plants. Several genes, which might dominate the fatty acid profile and seed oil content and have yet to be reported, were unveiled from the forward genetic studies. Our research provides valuable resources for functional genomics, elite crop breeding, and a good reference for high-throughput targeted mutagenesis in other polyploid plants.
最近利用 CRISPR-Cas9 系统构建的二倍体作物突变体文库为功能基因组学和作物育种提供了丰富的资源。然而，由于基因组的复杂性，在多倍体植物中实现大规模的靶向诱变仍然是一个巨大的挑战。在这里，我们展示了使用汇集的 CRISPR 文库在一个四倍体作物中实现全基因组靶向编辑的可行性。总共设计了 18414 个 sgRNA 来靶向 10480 个感兴趣的基因，随后获得了 1104 株含有 1088 个 sgRNA 的再生转基因植物。编辑检测结果表明，178 个基因中有 93 个被鉴定为突变，这代表了 52.2%的编辑效率。此外，我们还发现，在多倍体植物中，Cas9 介导的 DNA 切割倾向于发生在由同一单个 sgRNA 引导的所有靶位点。最后，我们展示了对各种性状进行反向遗传筛选的强大能力，这些植物是通过后基因型鉴定的。从正向遗传学研究中发现了几个可能主导脂肪酸组成和种子油含量的基因，这些基因尚未被报道。我们的研究为功能基因组学、优良作物育种提供了有价值的资源，也为其他多倍体植物的高通量靶向诱变提供了良好的参考。
