Regenerative Biology Research Laboratory, Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey; Koc University, Istanbul, Turkey.
Regenerative Biology Research Laboratory, Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey; Department of Medical Biology and Genetics, Faculty of Medicine, Ondokuz Mayis University, Samsun, Turkey.
Gene. 2020 Apr 15;734:144398. doi: 10.1016/j.gene.2020.144398. Epub 2020 Jan 24.
Recent developments in gene editing technology have enabled scientists to modify DNA sequence by using engineered endonucleases. These gene editing tools are promising candidates for clinical applications, especially for treatment of inherited disorders like sickle cell disease (SCD). SCD is caused by a point mutation in human β-globin gene (HBB). Clinical strategies have demonstrated substantial success, however there is not any permanent cure for SCD available. CRISPR/Cas9 platform uses a single endonuclease and a single guide RNA (gRNA) to induce sequence-specific DNA double strand break (DSB). When this accompanies a repair template, it allows repairing the mutated gene. In this study, it was aimed to target HBB gene via CRISPR/Cas9 genome editing tool to introduce nucleotide alterations for efficient genome editing and correction of point mutations causing SCD in human cell line, by Homology Directed Repair (HDR). We have achieved to induce target specific nucleotide changes on HBB gene in the locus of mutation causing SCD. The effect of on-target activity of bone fide standard gRNA and newly developed longer gRNA were examined. It is observed that longer gRNA has higher affinity to target DNA while having the same performance for targeting and Cas9 induced DSBs. HDR mechanism was triggered by co-delivery of donor DNA repair templates in circular plasmid form. In conclusion, we have suggested methodological pipeline for efficient targeting with higher affinity to target DNA and generating desired modifications on HBB gene.
基因编辑技术的最新进展使科学家能够通过使用工程内切酶来修饰 DNA 序列。这些基因编辑工具是临床应用的有前途的候选者，特别是对于治疗镰状细胞病（SCD）等遗传性疾病。SCD 是由人类β-球蛋白基因（HBB）中的一个点突变引起的。临床策略已经取得了巨大的成功，但目前还没有针对 SCD 的永久性治疗方法。CRISPR/Cas9 平台使用单个内切酶和单个向导 RNA（gRNA）诱导序列特异性 DNA 双链断裂（DSB）。当与修复模板一起使用时，它允许修复突变基因。在这项研究中，我们旨在通过 CRISPR/Cas9 基因组编辑工具靶向 HBB 基因，通过同源定向修复（HDR）在人类细胞系中引入核苷酸改变，以实现高效的基因组编辑和纠正导致 SCD 的点突变。我们已经成功地在导致 SCD 的突变基因座上诱导了 HBB 基因的靶特异性核苷酸变化。检查了针对靶基因的真实标准 gRNA 和新开发的更长 gRNA 的活性。结果表明，更长的 gRNA 与靶 DNA 的亲和力更高，而在靶向和 Cas9 诱导的 DSB 方面具有相同的性能。HDR 机制是通过共递送环形质粒形式的供体 DNA 修复模板触发的。总之，我们提出了一种高效靶向、与靶 DNA 具有更高亲和力并在 HBB 基因上产生所需修饰的方法学策略。
