Ishibashi Kazuhiro, Sukegawa Satoru, Endo Masaki, Hara Naho, Nureki Osamu, Saika Hiroaki, Toki Seiichi
Division of Plant Molecular Regulation Research, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Japan.
Division of Crop Genome Editing Research, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Japan.
Front Plant Sci. 2024 Sep 10;15:1454554. doi: 10.3389/fpls.2024.1454554. eCollection 2024.
Because virus vectors can spread systemically autonomously, they are powerful vehicles with which to deliver genome-editing tools into plant cells. Indeed, a vector based on a positive-strand RNA virus, potato virus X (PVX), harboring SpCas9 and its single guide RNA (sgRNA), achieved targeted mutagenesis in inoculated leaves of . However, the large size of the gene makes it unstable in the PVX vector, hampering the introduction of mutations in systemic leaves. Smaller Cas variants are promising tools for virus vector-mediated genome editing; however, they exhibit far lower nuclease activity than SpCas9. Recently, AsCas12f, one of the smallest known Cas proteins so far (one-third the size of SpCas9), was engineered to improve genome-editing activity dramatically. Here, we first confirmed that engineered AsCas12f variants including I123Y/D195K/D208R/V232A exhibited enhanced genome-editing frequencies in rice. Then, a PVX vector harboring this AsCas12f variant was inoculated into leaves by agroinfiltration. Remarkably, and unlike with PVX-SpCas9, highly efficient genome editing was achieved, not only in PVX-AsCas12f-inoculated leaves but also in leaves above the inoculated leaf (fourth to sixth upper leaves). Moreover, genome-edited shoots regenerated from systemic leaves were obtained at a rate of >60%, enabling foreign DNA-free genome editing. Taken together, our results demonstrate that AsCas12f is small enough to be maintained in the PVX vector during systemic infection in and that engineered AsCas12f offers advantages over SpCas9 for plant genome editing using virus vectors.
由于病毒载体能够自主进行全身传播，因此它们是将基因组编辑工具导入植物细胞的强大载体。事实上，一种基于正链RNA病毒——马铃薯X病毒（PVX）构建的载体，携带SpCas9及其单导向RNA（sgRNA），在接种的叶片中实现了靶向诱变。然而， 基因的大尺寸使其在PVX载体中不稳定，阻碍了在系统叶片中引入突变。较小的Cas变体是病毒载体介导的基因组编辑的有前景的工具；然而，它们的核酸酶活性远低于SpCas9。最近，AsCas12f是目前已知最小的Cas蛋白之一（大小为SpCas9的三分之一），经过改造后显著提高了基因组编辑活性。在这里，我们首先证实，包括I123Y/D195K/D208R/V232A在内的工程化AsCas12f变体在水稻中表现出更高的基因组编辑频率。然后，通过农杆菌浸润将携带这种AsCas12f变体的PVX载体接种到 叶片中。值得注意的是，与PVX-SpCas9不同，不仅在接种了PVX-AsCas12f的叶片中，而且在接种叶片上方的叶片（上部第四至第六片叶）中都实现了高效的基因组编辑。此外，从系统叶片再生出的基因组编辑芽的获得率>60%，实现了无外源DNA的基因组编辑。综上所述，我们的结果表明，AsCas12f足够小，能够在 全身感染期间在PVX载体中维持，并且工程化的AsCas12f在使用病毒载体进行植物基因组编辑方面比SpCas9具有优势。
