Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus
Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus
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DOI：
10.1111/tpj.13078
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发表时间：
2015-12-01
期刊：
影响因子：
7.2
通讯作者：
Puchta, Holger
中科院分区：
文献类型：
--
作者：
Steinert, Jeannette;Schiml, Simon;Puchta, Holger
聚类规则间隔短回文重复序列(CRISPR)/化脓性链球菌（SpCas9） Cas系统的应用正在给植物基因组工程带来革命性的变化。然而，合成植物生物学将需要对基因组和转录组进行更复杂的操作。在同一细胞内同时寻址具有独立酶活性的不同特定基因组位点是解决这一问题的关键。这些方法可以通过适应CRISPR/Cas系统的其他细菌同源物用于植物细胞来实现。在这里，我们发现来自嗜热链球菌（St1Cas9）和金黄色葡萄球菌（SaCas9）的密码子优化的Cas9同源物都可以用于诱导模式植物拟南芥中容易出错的非同源末端连接介导的靶向诱变，其频率至少与之前报道的化脓性链球菌CRISPR/Cas系统相当。在st1cas9和sacas9为基础的系统中，ADH1基因诱导的靶向突变在高频率下具有稳定的遗传。我们还能够证明SaCas9和SpCas9蛋白仅在其物种特异性单导rna存在时通过诱导双链断裂来增强同源重组。这些蛋白不容易受到异源sgRNA表达结构的种间干扰。因此，化脓性葡萄球菌和金黄色葡萄球菌的CRISPR/Cas系统应该适合同时处理同一植物细胞中具有不同酶活性的不同序列基序。
The application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system of Streptococcus pyogenes (SpCas9) is currently revolutionizing genome engineering in plants. However, synthetic plant biology will require more complex manipulations of genomes and transcriptomes. The simultaneous addressing of different specific genomic sites with independent enzyme activities within the same cell is a key to this issue. Such approaches can be achieved by the adaptation of additional bacterial orthologues of the CRISPR/Cas system for use in plant cells. Here, we show that codon-optimised Cas9 orthologues from Streptococcus thermophilus (St1Cas9) and Staphylococcus aureus (SaCas9) can both be used to induce error-prone non-homologous end-joining-mediated targeted mutagenesis in the model plant Arabidopsis thaliana at frequencies at least comparable to those that have previously been reported for the S. pyogenes CRISPR/Cas system. Stable inheritance of the induced targeted mutations of the ADH1 gene was demonstrated for both St1Cas9-and SaCas9-based systems at high frequencies. We were also able to demonstrate that the SaCas9 and SpCas9 proteins enhance homologous recombination via the induction of double-strand breaks only in the presence of their species-specific single guide (sg) RNAs. These proteins are not prone to inter-species interference with heterologous sgRNA expression constructs. Thus, the CRISPR/Cas systems of S. pyogenes and S. aureus should be appropriate for simultaneously addressing different sequence motifs with different enzyme activities in the same plant cell.