Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli
Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli
复制标题
DOI：
10.1016/j.fgb.2018.01.004
复制
发表时间：
2018-06-01
影响因子：
3
通讯作者：
Mortensen, Uffe H.
中科院分区：
文献类型：
--
作者：
Nodvig, Christina S.;Hoof, Jakob B.;Mortensen, Uffe H.
CRISPR-Cas9技术正在革新真菌基因编辑。本研究表明，Cas9/sgRNA介导的DNA双链断裂（DSBs）的存活依赖于非同源末端连接、NHEJ和DNA修复途径，并利用这一观察结果开发了一种工具——TAPE，以评估中性曲霉（Aspergillus nidulans）的原间隔器效率。此外，我们发现在缺乏NHEJ的菌株中，可以进行高效的无标记基因靶向。事实上，我们发现即使是单链寡核苷酸也能有效地作为A. nidulans、A. niger和A. oryzae中特定Cas9/sgRNA诱导的DNA dsb的修复模板，这表明这种类型的修复可能在丝状真菌中广泛存在。重要的是，我们证明了通过使用单链寡核苷酸进行CRISPR-Cas9介导的基因编辑，可以以接近100%的效率引入特定的点突变和基因缺失。该系统的效率要求多路复用，我们设计了一个基于聚合酶III启动子和tRNA间隔子的载体系统，具有递送Cas9和多个sgrna的能力。我们证明了在一次转化实验中以非常高的效率引入两个点突变和一个基因插入是可能的。我们的系统兼容未来的高通量基因编辑实验。
CRISPR-Cas9 technologies are revolutionizing fungal gene editing. Here we show that survival of specific Cas9/sgRNA mediated DNA double strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool, TAPE, to assess protospacer efficiency in Aspergillus nidulans. Moreover, we show that in NHEJ deficient strains, highly efficient marker-free gene targeting can be performed. Indeed, we show that even single-stranded oligo nucleotides efficiently work as repair templates of specific Cas9/sgRNA induced DNA DSBs in A. nidulans, A. niger, and in A. oryzae indicating that this type of repair may be wide-spread in filamentous fungi. Importantly, we demonstrate that by using single stranded oligo nucleotides for CRISPR-Cas9 mediated gene editing it is possible to introduce specific point mutations as well gene deletions at efficiencies approaching 100%. The efficiency of the system invites for multiplexing and we have designed a vector system with the capacity of delivering Cas9 and multiple sgRNAs based on polymerase III promoters and tRNA spacers. We show that it is possible to introduce two point mutations and one gene insertion in one transformation experiment with a very high efficiency. Our system is compatible with future high-throughput gene-editing experiments.