基于CRISPR/dCas9的新一代丝状真菌基因表达调控技术构建及其在蛋白质合成分泌途径研究中的应用

Upon endoplasmic reticulum stress, cells activate a series of complementary adaptive mechanisms to cope with protein-folding alteration, which together are known as the unfolded protein response (UPR). Although several aspects of ER stress response and the UPR have been intensively unraveled using the Saccharomyces cerevisiae model, however, the detailed molecular mechanism of UPR signaling in filamentous fungi, which are capable of much higher protein production levels such as lignocellulase, is still not clear. In the recent research, by using high-throughput mRNA sequencing technology (RNA-Seq) coupled to genetic screens, we undertook a systematic investigation of this process in the filamentous fungus Neurospora crassa using genome-wide transcriptional analysis coupled with mutant screening. Based on our recently successfully developed CRISPR/Cas9 system in the thermophilic filamentous fungi M. thermophila and M. heterothallica, in this project, we will construct complex synthetic transcriptional platform with CRISPR/dCas9 RNA scaffolds. By extending sgRNAs to include effectors protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. Moreover, this CRISPRi/a platform can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. We apply this approach to flexibly redirect the expression of 10 UPR target genes involved in lignocellulase secretion which were depend on the IRE-1 and Hac-1 in Neurospora crassa. Using this transcriptional-engineering tool, multiple strains exhibiting pronounced hyper or lower cellulase production will be generated. We will subsequently integrate these data with transcriptional and proteomic profiling. Finally, based on analysis of ChIP-sequencing、EMSA and Co-IP, we aim to dissect the comprehensive characterization of these 10 targets and uncover different interaction of theUbx-8, Der-1 and GRP78 during cellulase synthesis and secretion. Finally, in this project, we will develop the efficient CRISPR/dCas9 systems for genome editing and transcriptional regulation in the thermophilic species M. thermophila. By using this CRISPR/dCas9 system, up to seven genes involved in the cellulose production pathway will be simultaneously regulated in a one-step transformation. The CRISPR/dCas9 system developed in this project will accelerate genome-wide metabolic engineering of thermophilic fungal Myceliophthora species for the production of lignocellulases and bio-based fuels and chemicals. This project will enable systematic delineation of the complex machinery of the UPR and functional dissecting of the UPR target genes affecting ER homeostasis during in secretary pathway and would also provide rich biological insights for industrially technological cellulases production.

蛋白质合成分泌过程中的非折叠蛋白响应（UPR）是真核生物非常重要的基本生命过程。本研究将利用课题组前期已建立的丝状真菌CRISPR/Cas9基因编辑系统，构建丝状真菌新一代基因表达调控技术CRISPRi/a；其次，结合课题组前期挖掘影响蛋白分泌的10个新的UPR通路关键基因,利用CRISPRi/a在粗糙脉孢菌中对这些靶基因表达进行精细调控，通过鉴定系列组合突变株的基因表达、蛋白分泌、内质网形态和UPR程度等性状，综合利用转录组和分泌蛋白组、透射电镜、EMSA及Co-IP等，分析这些基因在丝状真菌蛋白分泌途径中的分子功能，进一步解析蛋白合成分泌中UPR应答的深层分子机制；同时，利用CRISPR编辑、调控技术和UPR研究最新发现，开展模式真菌粗糙脉孢菌和工业真菌嗜热毁丝霉UPR响应的比较研究。本课题的开展，对全面理解丝状真菌蛋白合成分泌中UPR应答的分子机理和工业真菌的遗传改造都具有重要意义。

本课题以嗜热毁丝霉为体系，研发构建了丝状真菌新型基因组编辑技术CRISPR-Cas12a系统以及新一代基因表达调控技术CRISPR-dCas9体系，结合前期挖掘的UPR通路的新靶标基因，利用构建的CRISPR编辑和调控技术，重点开展了模式真菌−粗糙脉孢菌和工业真菌−嗜热毁丝霉蛋白合成分泌调控机制的比较研究。课题已经按照计划进行，首先，基于V型Cas12a核酸酶构建了丝状真菌新型基因组编辑体系，通过Marker基因的回收和交替使用，构建了丝状真菌CRISPR-Cas9/Cas12a介导的Marker recycling基因编辑技术体系（CRISPR-Cas-assisted marker recycling technology），将其命名为Camr technology；其次，构建了基于dCas9的丝状真菌基因表达调控技术CRISPRi和CRISPRa，对UPR通路关键靶标转录因子进行了组合编辑和基因表达调控，构建了双、多基因转录抑制或激活的系列突变菌株IM-2、AM-2、AM-3和IM-9，实现靶向调控多基因的表达水平；第三，在粗糙脉孢菌和嗜热毁丝霉中，系统解析了参与纤维素酶表达分泌过程的一个新的关键转录因子CLR-4的分子功能，进而开展了粗糙脉孢菌和嗜热毁丝霉纤维素酶合成分泌途径调控机制的比较研究，结果表明CLR-4参与调控细胞生物量合成、纤维素酶产酶水平和酶活力，而且CLR-4在丝状真菌中的功能是保守的，CLR-4基因功能的解析有助于对丝状真菌蛋白分泌途径的认识，为纤维素酶高产菌的进一步改造提升提供了新思路；第四，利用构建的CRISPR编辑系统，结合UPR通路关键靶标基因cre-1，res-1、gh1-1、alp-1，rca-1、hcr-1、ap-3、prk-6、ubx-8和tah-1，人工重构嗜热毁丝霉蛋白合成分泌途径，获得了纤维素酶高产菌株M9和M11，同时也构建了嗜热毁丝霉异源糖化酶蛋白表达系统。课题执行期间，发表文章6篇，申请中国专利4项，联合培养研究生3名，完成了课题研究目标。