Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9.
Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9.
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DOI：
10.1038/srep26871
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发表时间：
2016-05-26
影响因子：
4.6
通讯作者：
Toki S
中科院分区：
文献类型：
--
作者：
Kaya H;Mikami M;Endo A;Endo M;Toki S
CRISPR/Cas9系统是植物基因组编辑的有效和方便的工具。来源于酿脓链球菌（Sp）的Cas9核酸酶通常用于该系统中。最近，金黄色葡萄球菌Cas9（SaCas 9）介导的基因组编辑在人类细胞和拟南芥中被报道。因为SaCas 9（1053 a.a.）小于SpCas 9（1368 a.a.），SaCas 9在递送和表达Cas9蛋白方面具有显著优势，特别是使用病毒载体。由于SaCas 9（5′-NNGRRT-3′）的前间区序列邻近基序（PAM）序列不同于SpCas 9（5′-NGG-3′）的前间区序列邻近基序（PAM）序列，因此使用这种替代Cas9核酸酶可以扩大CRISPR/Cas9系统的潜在切割靶位点处的选择性。在这里，我们表明SaCas 9可以对烟草和水稻中的靶序列进行突变，其效率与SpCas 9相似。我们还分析了在SaCas 9 PAM的第6位处对“T”的碱基偏好。非典型PAM（5′-NNGRRV-3′）对靶序列的定点突变效率远低于典型PAM（5′-NNGRRT-3′）。SaCas 9识别的靶序列的长度比SpCas 9识别的靶序列长一个或两个核苷酸。总之，我们的结果表明SaCas 9具有比SpCas 9更高的序列识别能力，并且可用于减少作物中的脱靶突变。
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.