Abstract：
The methylotrophic yeast Komagataella phaffii is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant \"dead\" MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research. CRISPRi utilizing dMAD7 does not introduce double-strand breaks but only binds to the DNA to regulate gene expression. Here, we report the first use of dMAD7 in K. phaffii to regulate the expression of the enhanced green fluorescent protein (eGFP). A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. Integration loci/events of investigated strains were assessed through whole genome sequencing. Additionally, RNA-sequencing experiments corroborated the whole genome sequencing results and showed a significantly reduced expression of eGFP in strains containing a dMAD7 plasmid, among others. Our findings conclusively demonstrate the utility of dMAD7 in K. phaffii through successfully regulating eGFP expression.
摘要：
甲基营养酵母Komagataellaphafii是用于重组蛋白的制药和生物技术生产的流行宿主系统。CRISPR-Cas9及其衍生的CRISPR干扰(CRISPRi)提供了进一步增强和利用该宿主的全部能力的有希望的途径。MAD7及其催化无活性变体“死”MAD7（dMAD7）代表了已建立的CRISPR-Cas9系统的有趣替代品，可免费用于工业和学术研究。利用dMAD7的CRISPRi不引入双链断裂,而是仅与DNA结合以调节基因表达。这里,我们报道了在K.phafii中首次使用dMAD7来调节增强型绿色荧光蛋白（eGFP）的表达。在使用dMAD7质粒的随机整合实验中实现eGFP荧光水平的降低(高达88%)。通过全基因组测序评估所研究菌株的整合基因座/事件。此外,RNA测序实验证实了全基因组测序结果，并显示含有dMAD7质粒的菌株中eGFP的表达显着降低，在其他人中。我们的发现最终证明了dMAD7通过成功调节eGFP表达在K.phafii中的实用性。
