【作者】 张鑫；

【导师】 荣知立；

【作者基本信息】 南方医科大学 ， 细胞生物学， 2021， 博士

【摘要】 基因编辑系统通常依赖于诱导DNA双链断裂(DSBs),然而由DSBs引起的脱靶位点的突变可能会产生有害的影响,从而限制其在临床治疗中的应用。最近CRISPR/Cas9系统被重新编辑并应用于基因的激活,它可以在天然染色体环境内对特定靶点进行基因激活而不产生DSBs,是一种很有潜力的治疗策略。目前,基于SpCas9已经开发多种基因激活系统,但是由于其原间隔子相邻基序(PAM)的限制、较高的脱靶效应、大体积、免疫原性等,使其在体内外的应用存在一些局限性。因此,我们仍需要构建不同类型的CRISPR激活系统,为在体内体外实现高效的基因激活提供新的可选择的方式。LbCpf1是一种2类V型CRISPR系统,不同于SpCas9识别5’-NGG的PAM序列,它可以识别5’-TTTV(V代表A,C或G)的PAM序列,并具有高特异性。我们将LbCpf1的核酸酶结构域的关键氨基酸进行突变,并结合组蛋白乙酰转移酶p300,构建dLbCpf1-p300激活系统。在gRNA的引导下,融合蛋白dLbCpf1-p300会与靶位点结合,并通过促进启动子和增强子区域组蛋白的乙酰化,使染色质发生重构,从而增强基因表达。同时,我们也将dLbCpf1突变体与SunTag系统结合,利用SunTag系统的中重复多肽GCN4和相应单链可变区抗体(scFv)特异性的结合,将融合蛋白ScFv-VP64招募到靶位点,使转录激活因子VP64在启动子区域富集,从而增强转录。通过这两种方式,我们基于LbCpf1构建了高特异性的转录激活系统,成功在多种细胞中诱导靶基因高效的表达,拓展了 CRISPR激活系统的应用范围。CjCas9是目前发现的最小Cas9同源体,与SpCas9相比,它具有不同的PAM识别序列,以及较小体积(984个氨基酸,而SpCas9为1,368个氨基酸)。因此我们将CjCas9与强效转录激活因子VPR结合,构建一系列转录激活系统。其中,dCjCas9-SunTag-VPR系统可以强效的激活基因的表达;CjCas9-VPR系统可以正交性诱导基因的编辑和激活;而基于VPR-dCjCas9系统进一步缩减和优化后形成的小型高效的激活体系(命名为miniCAFE),在基因插入细胞系中,单个拷贝miniCAFE即可在单条gRNA的引导下有效的激活靶基因的表达。同时,由于其小体积的优势,我们可以将miniCAFE和gRNA包装在一个AAV载体中进行体内传递,并成功激活肝脏中Fgf21的表达,调节成年小鼠的能量代谢。因此,miniCAFE为体内诱导基因的表达提供了一种新的方法。综合而言,我们基于LbCpf1和CjCas9构建了多种转录激活系统,并由于PAM的不同、LbCpf1的高特异性、CjCas9的小体积以及免疫原性,这些系统各有优势,为基因激活系统在体内外的应用提供了有力的补充,对人类疾病的治疗具有巨大的潜力。更多还原

【Abstract】 Genomic editing systems generally rely on the induction of DNA double-strand breaks(DSBs).However,off-target mutations caused by DSBs may have harmful effects,which limits their applications in clinical therapy.CRISPR/Cas9 system has recently been modified and applied to gene activation,which induces gene expression of target sites in the natural chromosomal environment without inducing DSBs,and is a promising therapeutic strategy.At present,a variety of gene activation systems based on SpCas9 have been developed.But due to the limitations of its PAM,high off-target effect,large size and immunogenicity,there are some limitations in its application in vitro and in vivo.Therefore,we still need to develop different types of CRISPR activation systems to provide new and alternative approaches for efficient gene activation in vitro and in vivo.CRISPR/LbCpf1 is a class 2 type Ⅴ CRISPR system.Unlike SpCas9,which recognizes PAM sequences 5’-NGG,LbCpf1 recognizes PAM sequences 5’-TTTV(V represents A,C or G)with high specificity.Therefore,LbCpfl(LbCas12a)was deactivated via mutagenesis in the nuclease domain and fused p300 to construct dLbCpf1-p300 system.Under the guidance of gRNA,dLbCpfl-p300 fusion proteins bind to the target sites and promote the acetylation of histones in the promoter and enhancer regions,which results gene expression activation.Meanwhile,dLbCpf1 also combined with Suntag system,which consists of repeating peptide GCN4 and a scFv antibody-fusion protein.In dLbCpf1-SunTag system,the fusion protein SCFV-VP64 can be recruited to the target site by specific binding between GCN4 and scFv,which results the transcriptional activator VP64 to accumulate in the promoter region,thereby enhancing transcription.Through these two approaches,we constructed a highly specific dLbCpfl-based transcriptional activation system,which successfully induced the highly efficient expression of target genes in a variety of cells,expanded the application range of CRISPR activation system.CjCas9 is the smallest Cas9 homologue with a different PAM sequence and a smaller size(984 amino acids compared to 1,368 amino acids of SpCas9).Therefore,we constructed a series of transcriptional activation systems combined with the minimal CjCas9(derived from Campylobacter jejuni,984aa),and the potent transcriptional activator VPR.Among them,dCjCas9-SunTag-VPR system can strongly activate gene expression;The CjCas9-VPR system can orthogonally induce gene editing and activation.More importantly,a small and efficient activation system(named MiniCAFE),Optimization and minimized based on dCjCas9-VPR,can efectively activate the expression of target genes by combining a single copy of miniCAFE with a single gRNA.At the same time,due to its small size,we could package miniCAFE and gRNA in an AAV carrier for delivery,and successfully activated the expression of FGF21 in the liver to regulate the energy metabolism of adult mice.Therefore,miniCAFE provides a powerful way to induce gene expression in vivo.In conclusion,we have constructed a variety of transcriptional activation systems based on LbCpfl and CjCas9.Due to the characteristics of these two systems,such as different PAM,high specificity of LbCpfl,small size of CjCas9 and immunogenicity,these systems have different advantages and provide a powerful supplement for the application of gene activation systems in vitro and in vivo,and has great potential for the treatment of human diseases.更多还原