The Construction Of CRISPR-dCas9/Cas13a-Based Systems For Transcriptional And Post-Transcriptional Regulation And Its Application

Precise and carefully orchestrated set of steps that depend on the proper spatial and temporal experession of genes enables faithful execution of biological processes such as development,proliferation,apoptosis,aging,and differentiation.Therefore,deregulation of gene expression may result in disease.In eukaryotes,expression of genes can be regulated at several steps,including transcription initiation and elongation,and RNA processing,transport,translation,and stability.Thus,the ability to regulate these processes enables to manipulate and interfere with gene expression,which may help to understand the complex functions of gene network,study disease pathogenesis and develop therapeutic interventions.The discovery of CRISPR system have provided a powerfun tool for controlling gene expression.In this paper,we focus on the construction of CRISPR-based systems and its application for transcriptional activation and post-transcriptional regulation.Target gene activation or repression can be realized by fusing transcriptional effector domains to nuclease-null dCas9 protein.Furthermore,multi-module dCas9 systems have been developed for controlling dCas9-based gene regulation by introducing various light-or small-molecule-inducible dimerrizing domains into the CRISPR-dCas9 system.However,there is still a need for a new module to achieve more robust and flexible dCas9-based transcription control.Therefore,in chapter 2,we construct a dCas9-based transcriptional regulation system mediated by a separate RNA module that can recruit multiple effector domains through RNA-protein interaction.Using this new system,we further improved the efficiency of gene activation.As a separate component,the RNA module acts as a bridge between the dCas9 and effector domains and thus can be engineered to recruit desired copies of effector domians without disturbing the sgRNA structure.Subsequently,we generated a circuit responding to endogenous microRNAs by introducing a protein competing with dCas9 fusion for the separate RNA module,and achieved cancer cell identification in cell mixtures in chapter 3.These work provide a flexible platform for efficient and controllable gene regulation based on CRISPR-dCas9.In addition to transcriptional regulation,CRISPR-dCas9 have been used in many important fields such as base editing,genome modification,and chromosome imaging.To effectively control dCas9 based applications,protein-based inhibitor of Cas9,anti-CRISPR,have been used to realize efficient inhibition of Cas9 systems.In contrast to genetic methods,small-molecule inhibitors show fast kinetics,which means more precise control.In addition,small-molecule inhibitors can be cell permeable,reversible,proteolytically stable,and non-immunogenic.In chapter 4,we construct a dCas9-based transcription activation system that depends on protein-protein interaction between ALK4 and FKBP12,which is can be disrupted by the FK506 compound.In our system,the small molecule FK506 act as a brake that can inhibit dCas9-based transcription activation readily by targeting ALK4-FKBP12 interaction.We varied the dose and duration of action of FK506 and demonstrated that FK506 inhibits gene expression in a time-and dose-dependent manner.Therefore,this system offers a simple and versatile method for developing small-molecule inhibitors to control dCas9-based gene regulation.In addition,post-transcriptional regulation is also an efficient way to control gene expression.The discovery of CRISPR-Cas 13 provides a useful tool to target RNA and has been widely used in mammalian cells due to the higher efficiency of RNA knockdown and lower off-target effects relative to RNAi.In the commonly used form that Cas13 system is delivered,RNA dose not need to enter the nucleus,thus reducing the risk of insertion mutation.Meanwhile,mRNA is only transiently active,which will reduce the off-target effect and may be beneficial for applications where transient protein expression is required.In chapter 5,in order to study the efficiency of RNA-based Cas13 system in vivo,we synthesized Cas13 mRNA and various crRNA in vitro and delivered these RNA into cells.We found that varying the length of crRNA spacer and the structure of the direct repeat sequence could lead to different change of knockdown efficiency.This work provides a reference for the design and selection of crRNA when Cas13 is used for RNA therapy in the future.