■柑橘溃疡,由柠檬酸黄单胞菌亚种引起。citri(Xcc),是全世界范围内的毁灭性疾病。以前,我们成功地产生了抗溃疡的柑橘cv。T0代中的Hamlin线。这是通过使用含有Cas12a和一个crRNA的核糖核蛋白(RNP)编辑溃疡易感性基因来转化胚性原生质体来实现的。CsLOB1,这导致了小的indel。
■这里,我们用含有Cas12a和三个crRNA的RNP转化了Hamlin的胚发生原生质体。
■在10个无转基因基因组编辑品系中,在五个系中获得了长缺失。此外,在五个编辑的线中的三个中观察到倒位,这些线具有长缺失,但在任何具有短indel突变的编辑行中都没有,提示倒位可能需要长时间删除。当使用三种crRNA时,在10个编辑品系中的四个中观察到三个靶位点中的每一个的双等位基因突变,证明用Cas12a和三种crRNARNP转化胚发生的柑橘原生质体对于多重编辑是非常有效的。我们的分析揭示了编辑品系中不存在脱靶突变。这些cslob1突变品系具有抗溃疡性,并且在接种Xcc后没有观察到溃疡症状,并且与野生型植物相比,cslob1突变品系中的Xcc生长显著降低。
■放在一起,柑橘胚发生原生质体的RNP(Cas12a和三个crRNA)转化为高效的无转基因多重基因组编辑和长片段的缺失提供了有希望的解决方案。
UNASSIGNED: Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease worldwide. Previously, we successfully generated canker-resistant Citrus sinensis cv. Hamlin lines in the T0 generation. This was achieved through the transformation of embryogenic protoplasts using the ribonucleoprotein (RNP) containing Cas12a and one crRNA to edit the canker susceptibility gene, CsLOB1, which led to small indels.
UNASSIGNED: Here, we transformed embryogenic protoplasts of Hamlin with RNP containing Cas12a and three crRNAs.
UNASSIGNED: Among the 10 transgene-free genome-edited lines, long deletions were obtained in five lines. Additionally, inversions were observed in three of the five edited lines with long deletions, but not in any edited lines with short indel mutations, suggesting long deletions maybe required for inversions. Biallelic mutations were observed for each of the three target sites in four of the 10 edited lines when three crRNAs were used, demonstrating that transformation of embryogenic citrus protoplasts with Cas12a and three crRNAs RNP can be very efficient for multiplex editing. Our analysis revealed the absence of off-target mutations in the edited lines. These cslob1 mutant lines were canker- resistant and no canker symptoms were observed after inoculation with Xcc and Xcc growth was significantly reduced in the cslob1 mutant lines compared to the wild type plants.
UNASSIGNED: Taken together, RNP (Cas12a and three crRNAs) transformation of embryogenic protoplasts of citrus provides a promising solution for transgene-free multiplex genome editing with high efficiency and for deletion of long fragments.