【作者】 张涛；

【导师】 赖良学；

【作者基本信息】 吉林大学 ， 兽医学， 2024， 博士

【摘要】 CRISPR/Cas系统作为一种革命性的基因编辑技术,已经广泛应用于基础研究、基因治疗、核酸检测、诊断、成像和抗病毒等方面。CRISPR/Cas13系统是RNA介导的靶向单链RNA(ss RNA)的核酸酶,其具有pre-cr RNA加工活性和RNA靶向活性。Cas13蛋白的大小普遍在950AA-1300AA之间,相对较大。然而,由于腺相关病毒(AAV)的体内递送限制,开发用于基因治疗的CRISPR/Cas编辑系统仍然具有挑战性。因此,紧凑型Cas蛋白酶的开发迫在眉睫。目前,相关研究已经提出了几种用于Cas蛋白小型化的成功策略。主要包括:1)在宏基因组数据集中开发天然紧凑的Cas蛋白酶。然而,这些紧凑的Cas蛋白并不完全具有先前优化的Cas蛋白的优势。2)通过修改Cas酶的功能,去除不需要的功能结构域。但在这种策略中,Cas蛋白的DNA/RNA切割活性通常被消除,仅仅保留DNA/RNA结合活性。3)通过生成随机缺失突变体库来呈现Sp Cas9的整体缺失,使得突变体分别仅保留约72%和63%的Sp Cas9蛋白序列,DNA结合和切割活性相应下降。4)通过构建Cas13d直系同源物中保守性差的表面定位缺失变体,以最小的效率损失减小了蛋白大小,但该过程在很大程度上受到可用片段的限制,无法满足Cas蛋白未来应用的小型化要求。由于蛋白小型化存在以上的问题,因此,需要开发新的用于基因治疗的小型化策略。基于以上问题,本研究旨在开发新的小型化策略以开发紧凑型的Cas蛋白,并利用小型化的Cas13蛋白构建相应的RNA编辑系统和进行基因治疗。首先,我们提出了一种基于蛋白质结构和Alpha Fld2的蛋白质小型化策略——IDC策略,其目的是最大限度地使蛋白质小型化,同时保留蛋白质功能。IDC策略是通过分析蛋白结构中蛋白-核酸相互作用、构象动力学和蛋白家族保守性而建立的。I代表"interaction"。Cas13蛋白在行使功能时,要经历从单一蛋白到与cr RNA结合形成二元复合物,再与target-RNA结合形成三元复合物的过程。在Cas13的整个功能发挥过程就主要围绕着RNA进行。因此,在蛋白结构上,与RNA发生相互作用的结合位点以及支撑结合位点的支架结构单元是至关重要的。D代表"Dynamic"。在Cas13蛋白功能行使过程中,各个功能结构域有不同程度的变构活动。所以还要分析在变构过程中各个结合位点和支架结构单元的变化。我们通过分析动态过程中的结合位点和支架结构单元,就能够对整个蛋白的结构进行排除式地删除,以此生成紧凑型的Cas13d蛋白。C代表"Conservation"。Cas13蛋白家族具有高度的结构保守性,因此以上“I”和“D”的分析可以应用于结构保守性高的蛋白家族。所以将以上的三个分析过程"Interaction","Dynamic"以及"Conservation"进行提炼,提出了蛋白小型化策略-IDC策略。其次,基于IDC策略和AlphaFold2预测结构,成功生成了三种紧凑型的Cas13d变体。三种mini-Cas13d变体具有完全的RNA结合和切割活性,在293T细胞中具有一致的敲低效率。此外,我们系统性的探究了mini-RfxCas13d的体内外功能活性。结果发现mini-RfxCas13d的活性功能不仅与RfxCas13d基本一致,且相较于RfxCas13d具有高保真活性,其脱靶率显著降低。随后,为了扩大IDC策略的应用范围,我们针对两种应用广泛的Cas13b蛋白生成了mini-Cas13b变体。两种mini-Cas13b变体保留了与野生型蛋白酶一致的酶活性。最后,为了验证mini-Cas13蛋白是否能够应用于RNA碱基编辑和基因治疗。我们将mini-RfxCas13d蛋白与ADAR2的脱氨酶结构域融合构建了一个RNA碱基编辑器mini-Vx,其具有与Vx一致的A-I的编辑能力,并且基本不会引起转录组中广泛的碱基突变。最后,通过将mini-RfxCas13d和cr RNA共同包装至AAV病毒中(腺相关病毒),成功降低了小鼠肝脏中Pcsk9的表达水平。这表明mini-RfxCas13d蛋白能够在动物体内成功的发挥活性,可以用于基因治疗。综上所述,本研究通过AlphaFold2预测和分析Cas13蛋白结构,成功地提出了IDC策略,利用IDC策略对Cas13b和Cas13d蛋白家族进行优化,生成了5种具有完全RNA结合和切割活性的紧凑型变体。同时,发现mini-RfxCas13d是一种高保真活性Cas蛋白酶,并且能够进行RNA碱基编辑和用于基因治疗。这一研究为未来基因治疗中紧凑型蛋白的开发和应用提供了新的策略和工具。更多还原

【Abstract】 The CRISPR-Cas system has been developed to be a revolutionary gene-editing technology that has been developed for basic research,therapeutics,nucleic acid detection,diagnostics,imaging,and antiviral applications.The CRISPR/Cas13 system is an RNA mediated nuclease targeting single stranded RNA(ss RNA),which exhibits pre cr RNA processing activity and RNA targeting activity.The size of Cas13 protein is generally between 950 AA and 1300 AA,which is relatively large.However,the CRISPR-Cas editing systems for the therapeutic applications remain challenging due to the in vivo delivery constraints of adeno-associated virus(AAVs).Therefore,the development of compact Cas proteases is urgent.At present,several successful strategies for miniaturizing Cas proteins have been proposed in relevant studies.This mainly includes: 1)developing naturally compact Cas proteases in the metagenomic dataset.However,these compact proteins don’t completely recapitulate the advantages of previously optimized Cas proteins.2)the functionalities of Cas enzymes are modified by removing unnecessary functional domains.However,in this strategy,the Cas DNA-/RNA-cleavage activity is commonly eliminated,whereas its DNA-/RNA binding activity is preserved.3)By generating a random deletion mutant library to present the overall deletion of Sp Cas9,the mutants only retained about 72% and 63% of the Sp Cas9 protein sequence,respectively,with a corresponding decrease in DNA binding and cleavage activity.4)By constructing surface-localized deletion variants of poorly conserved Cas13 d orthologs allow protein size reduction with minimal loss of efficiency,this process is largely restricted by the available fragments being unable to meet the miniaturization requirements of a Cas protein for future applications.Due to the aforementioned challenges,it is necessary to develop a miniaturization strategy for gene therapy.Based on the above issues,this study aims to develop new miniaturization strategies to develop compact Cas proteins,and construct corresponding RNA editing systems and gene therapy using miniaturized Cas13 proteins.Firstly,we propose an IDC strategy for protein miniaturization based on protein structure and AlphaFold2,with the goal of maximizing protein miniaturization while preserving protein function.The IDC strategy is established by analyzing protein nucleic acid interactions,conformational dynamics,and protein family conservation in protein structure."I" represents "interaction".When Cas13 protein functions,it undergoes a process from a single protein to binding with cr RNA to form a binary complex,and then binding with target RNA to form a ternary complex.The entire functional process of Cas13 mainly revolves around RNA.Therefore,in terms of protein structure,the binding sites that interact with RNA and the scaffold structural units that support the binding sites are crucial."D" represents "Dynamic".During the functional exercise of Cas13 protein,various functional domains exhibit varying degrees of conformational activity.Thus,it is necessary to analyze the changes in various binding sites and scaffold structural units during the transformation process.By analyzing the binding sites and scaffold structural units in the dynamic process,we can exclude the entire protein structure and generate a compact Cas13 d protein."C" represents "Conservation".The Cas13 protein family has a high degree of structural conservation,so the analysis of "I" and "D" above can be applied to protein families with high structural conservation.So,the above three analysis processes "Interaction","Dynamic",and "Conservation" were refined,and a protein miniaturization strategy-IDC strategy was proposed.Secondly,based on the IDC strategy and AlphaFold2 prediction structure,three compact Cas13 d variants were successfully generated.Three mini Cas13 d variants exhibit complete RNA binding and cleavage activity,with consistent knockdown efficiency in 293 T cells.Subsequently,in order to expand the application scope of the IDC strategy,we generated mini Cas13 b variants targeting two widely used Cas13 b proteins.Two variants of mini Cas13 b retained enzyme activity comparable to wildtype proteases.In addition,we systematically investigated the in vivo and in vitro functional activity of mini RfxCas13 d.The results showed that the active function of mini RfxCas13 d was not only similar to that of RfxCas13 d,but also had high fidelity activity compared to RfxCas13 d,and its off-target rate was significantly reduced.Further utilizing the mini RfxCas13 d protein,an RNA base editor mini Vx was constructed,which has the same A-I editing ability as Vx and does not cause widespread base mutations in the transcriptome.Finally,by co packaging mini RfxCas13 d and cr RNA into AAV virus(adeno-associated virus),the expression level of Pcsk9 in mouse liver was successfully reduced.This indicates that the mini RfxCas13 d protein can successfully exert its activity in animals and can be used for gene therapy.In summary,this study successfully proposed the IDC strategy by analyzing AlphaFold2 to predict and analyze the structure of Cas13 protein,and used the IDC strategy to generate five compact variants with complete RNA binding and cleavage activity for Cas13 b and Cas13 d families.Meanwhile,it was found that mini RfxCas13 d is a high-fidelity active Cas protease that can be used for RNA editing and gene therapy.This study provides new strategies and tools for the development and application of compact proteins in future gene therapy.更多还原