【作者】 龙洁；

【导师】 姚少华；

【作者基本信息】 四川大学 ， 细胞生物学， 2021， 硕士

【摘要】 目的:单碱基编辑技术可以高效、精准的实现靶DNA的单个碱基的转换,但存在脱靶效应和受限于固定的编辑窗口等缺点,针对碱基编辑的脱靶效应和编辑窗口,我们开发一系列新型的碱基编辑工具以最大程度地减少脱靶效应和拓展编辑窗口。单碱基编辑技术通常由一个胞嘧啶脱氨酶或腺嘌呤脱氨酶融合单链催化活性的Cas9（n Cas9）而成,包括胞嘧啶碱基编辑器（CBE）和腺嘌呤碱基编辑器（ABE）。相对传统的CRISPR-Cas9介导的同源重组,碱基编辑技术大大提高了碱基替换的效率,由于不产生DNA双链断裂,其安全性也更高。目前,其广泛应用于基因功能研究、遗传疾病机制探究、基因治疗及作物改良等领域。尽管碱基编辑如此强大,但仍有不足之处。例如,碱基编辑存在DNA和RNA水平的脱靶效应,有些碱基编辑酶在体内过表达还会导致肿瘤发生,编辑活性不受控制的碱基编辑器大大限制了其临床应用的转化;此外,有些复杂疾病位点的窗口范围内具有多个碱基A或C,这促使我们积极开发新型碱基编辑工具用以实现精准的碱基替换和更多突变组合的编辑。本研究主要通过对碱基编辑器的优化和改造,构建了一系列新的碱基编辑工具,减少了脱靶编辑,缩小或扩大编辑窗口,为碱基编辑工具的临床应用提供了新的方案以及个性化编辑提供了更多选择。方法:1)通过生物学软件Py MOL对脱氨酶或SaCas9/sg RNA/DNA复合物的结构进行可视化分析。2)通过分子克隆技术、细胞转染、基因组提取、PCR扩增以及Sanger测序等技术对新型碱基编辑工具进行活性验证及筛选,并与原始版本的编辑器进行比较。3)进一步表征了新型碱基编辑工具的主要编辑模式,例如编辑窗口,序列偏好性,编辑纯度。4)通过融合核易位系统进一步优化了新型碱基编辑工具。5)通过脱靶编辑的检测对其进行安全性评价。6)根据编辑器的特点,探究了其在基因治疗的应用。结果:首先,我们开发了诱导型的CBE碱基编辑系统,以胞嘧啶脱氨酶A3A为模型构建了一系列诱导型碱基编辑器:1)通过将A3A分裂为失活的N端和C端后,分别融合至FRB和FKBP蛋白的方法,构建了split-A3A-BE3。其中,sA3A-BE3-85对雷帕霉素具有最好的响应。诱导时,平均编辑效率可达A3A-BE3的80%,无诱导时,几乎没有编辑活性。2)通过进一步优化诱导条件,发现其对雷帕霉素的诱导具有时间和剂量依赖性。3)进一步表征其编辑模式,发现sA3A-BE3-85的编辑窗口及序列偏好没有明显变化,而在编辑纯度方面,sA3A-BE3-85具有更高的C→T效率。4)我们检测到sA3A-BE3-85有低水平的背景编辑,为此我们通过在sA3A-BE3-85的N端融合核易位系统,将背景编辑降低至几乎不可检测的水平。5)我们还将这种方案应用到碱基编辑中另一个广泛使用的脱氨酶,APOBEC1,获得了编辑活性可控的s APOBEC1-BE3编辑器。另外,我们还开发了内部镶嵌的SaCas9碱基编辑器:1)通过将A3A嵌入Cas9内部的不同位置构建了Sa-CBE-N、SaCBE-125、SaCBE-269、SaCBE-593、SaCBE-693等编辑器。2)构建完成后进行活性验证,发现这种策略扩大或缩小了编辑窗口。尤其是SaCBE-693将窗口由原来的3～14拓宽至3～18;SaCBE-593虽然效率较低但同样拓宽了窗口（3～18）;相反,SaCBE-125将编辑窗口缩小至8～15位。3)基于以上发现,我们还构建了双碱基编辑工具,Sa-CABE-693。构建完成后进行活性验证,发现与N端融合脱氨酶的方案（Sa-CABE-N）相比,我们的方案具有更宽泛的编辑窗口,可编辑更多A&C的突变组合。4)我们进一步探究了Sa-CABE-693在基因治疗方面的潜力。通过构建BCL11A-sg RNA以靶向+58BCL11A增强子的GATA1结合基序,发现两种方案在A₆、C₈、A₉碱基的编辑效率基本一致,不同的是Sa-CABE-693在C₁₇能达到40%左右效率,约Sa-CABE-N的3倍。结论:1)诱导型碱基编辑系统可有效的控制脱氨酶活性,并且具有更高的编辑纯度,为及时控制碱基编辑引起的脱靶编辑提供了可行性方案,为碱基编辑的临床应用提供了新的思路。2)内部镶嵌的SaCas9碱基编辑器,大大扩展了编辑窗口,尤其是SaCBE-693将窗口拓宽了约3～4个碱基。3)Sa-CABE-693作为双碱基编辑工具不仅能同时编辑A&C碱基,同时与N端融合脱氨酶的方案相比,进一步扩宽了C→T的编辑范围,并且更有效的引入治疗β-血红蛋白病的突变。更多还原

【Abstract】 Objective:We aimed to develop a series of new base editing tools with minimizing off-target effects and expanding editing window.Single base editing techniques,including cytosine base editors（CBEs）and adenine base editors（ABEs）,usually consists of a cytosine deaminase or adenine deaminase fused with single-stranded catalytically active Cas9（n Cas9）.Compared with the traditional CRISPR-Cas9-mediated homologous recombination,the base editing technology greatly improves the efficiency of base substitution.Because it does not produce DNA double-strand breaks,it’s also much safer.At present,it is widely used in the fields of gene function research,genetic disease mechanism exploration,gene therapy and crop improvement.Although base editing is robust,there are still disadvantages.For example,the base editors can induce off-target editing at the DNA and RNA levels.The overexpression of deaminase can also lead to tumorigenesis.Base editors with uncontrolled editing activity greatly limit their clinical application.In addition,some complex disease sites have multiple bases A or C within the window,which prompts us to actively develop new base editing tools to achieve precise base substitution and editing of more mutation combinations.In this study,a series of new base editing tools were constructed through the optimization and transformation of the base editor,which reduced off-target editing,narrowed or expanded the editing window,and provided a new solution for the clinical application of base editing tools or provided more options for personalized editing.Methods:1)Intuitively analyze the structure of deaminase or SaCas9/sg RNA/DNA complex through the biology software Py MOL.2)Through molecular cloning technology,cell transfection,genome extraction,PCR amplification and Sanger sequencing and other molecular biology methods,the activity of the new base editing tool was verified and compared with the original editor.3)We further described the main editing modes of the new basic editing tools,such as the editing window,sequence preferences and editing purity.4)The new basic editing tool has been further optimized through the fusion nuclear translocation system.5)Conduct security assessments of these editors by testing off-target editing.6)According to the editor’s characteristics,explore its application in human disease animal models or gene therapy.Results:First,we developed an inducible base editing system,Split-A3A-BE3:1)The inducible bases were constructed by splitting A3A into the inactive N-terminal and C-terminal and then fused to the FRB and FKBP proteins respectively.We found that sA3A-BE3-85 had the best response to the induction of rapamycin.sA3A-BE3-85 has similar activity to the original version of the base editor under induction conditions,and its average editing efficiency can reach 80%compared with the full length,and there is little editing without induction.2)By further optimizing the induction conditions of sA3A-BE3-85,it is found that the induction of rapamycin is time-dependent and dose-dependent.3)The main editing mode was further characterized,and it was found that compared with A3A-BE3,the editing window and sequence preference of sA3A-BE3-85 did not change significantly,while in terms of editing purity,sA3A-BE3-85 has higher The C→T efficiency.4)We detected that sA3A-BE3-85 has a low level of background editing.For this reason,we reduced the background editing to an almost undetectable level by fusing the nuclear translocation system at the N-terminal of sA3A-BE3-85.5)We also applied this scheme to another widely used deaminase in base editing,rat APOBEC1,the result is similar to sA3A-BE3-85.In addition,we have also developed a base editor for embedding deaminase in the SaCas9 domain:1)By embedding A3A in different positions of the Cas9 domain,we have constructed editors such as Sa-CBE-N、SaCBE-125、SaCBE-269、SaCBE-593 and SaCBE-693.2)By testing the activity of these editors,we found that compared with the original base editor,they have a different editing window.SaCBE-693expands the window to 3～18,expanding about 3～4 bases;SaCBE-593shows a lower editing efficiency but also expanded the editing window（3～18）,in contrast,SaCBE-125 narrows the editing window from 3～14to 8～15.3)Based on the above findings,we also constructed a dual-deaminase CRISPR base editor,Sa-CABE-693.Compared with the N-terminal fusion deaminase solution,Sa-CABE-693 has a wider editing window and can edit more A&C mutation combinations.4)Based on this feature,we further verified the potential of Sa-CABE-693 in gene therapy.By constructing BCL11A-sg RNA to target the GATA1 binding motif of the+58 BCL11A enhancer,it was found that the editing efficiency of the two schemes at bases A₆,C₈,and A₉ was equivalent.The difference is that Sa-CABE-693 can reach 40%at C₁₇,about 3times that of Sa-CABE-N.Conclusion:1)The inducible base editing system can effectively control the deaminase activity and has higher editing purity.It provides a feasible solution for timely control of off-target effects caused by base editing,and provides a new idea for the clinical application of base editing.2)The base editor of deaminase is embedded in the SaCas9domain,which widen or narrow the editing window and provides a better solution for editing some complex sites.3)As a double-base editing tool,Sa-CABE-693 can not only edit A&C bases at the same time,but compared with the N-terminal fusion deaminase solution,it further broadens the editing range from C to T and is effective to introduce mutations for the treatment ofβ-hemoglobinopathy.更多还原