SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast.
SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast.
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DOI：
10.12688/wellcomeopenres.16405.1
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发表时间：
2020
影响因子：
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通讯作者：
Allshire RC
中科院分区：
文献类型：
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作者：
Torres-Garcia S;Di Pompeo L;Eivers L;Gaborieau B;White SA;Pidoux AL;Kanigowska P;Yaseen I;Cai Y;Allshire RC
CRISPR/Cas9系统允许无疤痕，无标记的基因组编辑。目前用于裂殖酵母裂殖酵母的CRISPR/Cas9系统依赖于繁琐且耗时的克隆程序来将特定的sgRNA靶序列引入表达Cas9的质粒中。此外，据报道，Cas9内切核酸酶在从强adh 1启动子组成型过表达时对裂殖酵母有毒。为了克服这些问题，我们开发了一种改进的系统SpEDIT，该系统使用针对S. SpEDIT系统表现出灵活的模块化设计，其中sgRNA与自切割丁型肝炎病毒（HDV）核酶的3'端融合，允许由RNA聚合酶III从tRNA基因序列驱动sgRNA盒的表达。包含BsaI型IIS限制性酶的位点位于GFP占位符的侧翼，使得能够用合成的sgRNA一步介导GFP的替换以用于表达。SpEDIT系统允许100%的诱变效率，当通过转化来自异步培养物的细胞在ade 6+或ura 4+基因中产生靶点突变体时，SpEDIT还允许以最小的努力获得插入、标记和缺失事件。同时编辑两个独立的非同源基因座也很容易实现。重要的是，与目前可用的S相比，SpEDIT系统显示出降低的毒性。pombe编辑系统。因此，SpEDIT提供了一种有效且用户友好的CRISPR/Cas9程序，显著改善了裂变酵母的基因组编辑工具箱。
The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong adh1 promoter. To overcome these problems we have developed an improved system, SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for S. pombe expressed from the medium strength adh15 promoter. The SpEDIT system exhibits a flexible modular design where the sgRNA is fused to the 3’ end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The SpEDIT system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the ade6 + or ura4 + genes by transformation of cells from asynchronous cultures. SpEDIT also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the SpEDIT system displayed reduced toxicity compared to currently available S. pombe editing systems. Thus, SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.