Kaya Hidetaka, Mikami Masafumi, Endo Akira, Endo Masaki, Toki Seiichi
Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Yokohama, Kanagawa 236 0027, Japan.
Sci Rep. 2016 May 26;6:26871. doi: 10.1038/srep26871.
The CRISPR/Cas9 system is an efficient and convenient tool for genome editing in plants. Cas9 nuclease derived from Streptococcus pyogenes (Sp) is commonly used in this system. Recently, Staphylococcus aureus Cas9 (SaCas9)-mediated genome editing was reported in human cells and Arabidopsis. Because SaCas9 (1053 a.a.) is smaller than SpCas9 (1368 a.a.), SaCas9 could have substantial advantages for delivering and expressing Cas9 protein, especially using virus vectors. Since the protospacer adjacent motif (PAM) sequence of SaCas9 (5'-NNGRRT-3') differs from that of SpCas9 (5'-NGG-3'), the use of this alternative Cas9 nuclease could expand the selectivity at potential cleavage target sites of the CRISPR/Cas9 system. Here we show that SaCas9 can mutagenize target sequences in tobacco and rice with efficiencies similar to those of SpCas9. We also analyzed the base preference for 'T' at the 6th position of the SaCas9 PAM. Targeted mutagenesis efficiencies in target sequences with non-canonical PAMs (5'-NNGRRV-3') were much lower than those with a canonical PAM (5'-NNGRRT-3'). The length of target sequence recognized by SaCas9 is one or two nucleotides longer than that recognized by SpCas9. Taken together, our results demonstrate that SaCas9 has higher sequence recognition capacity than SpCas9 and is useful for reducing off-target mutations in crop.
CRISPR/Cas9系统是植物基因组编辑中一种高效便捷的工具。该系统中常用的Cas9核酸酶来源于化脓性链球菌（Sp）。最近，有报道称金黄色葡萄球菌Cas9（SaCas9）介导的基因组编辑可应用于人类细胞和拟南芥中。由于SaCas9（1053个氨基酸）比SpCas9（1368个氨基酸）小，SaCas9在Cas9蛋白的传递和表达方面可能具有显著优势，尤其是在使用病毒载体时。由于SaCas9的前间隔序列临近基序（PAM）序列（5'-NNGRRT-3'）与SpCas9的不同（5'-NGG-3'），使用这种替代性Cas9核酸酶可以扩大CRISPR/Cas9系统潜在切割靶点的选择性。在此我们表明，SaCas9能够诱变烟草和水稻中的靶序列，其效率与SpCas9相似。我们还分析了SaCas9的PAM序列第6位对“T”的碱基偏好性。具有非典型PAM（5'-NNGRRV-3'）的靶序列中的靶向诱变效率远低于具有典型PAM（5'-NNGRRT-3'）的靶序列。SaCas9识别的靶序列长度比SpCas9识别的长1个或2个核苷酸。综上所述，我们的结果表明，SaCas9比SpCas9具有更高的序列识别能力，并且可用于减少作物中的脱靶突变。
