Comparison of the editing patterns and editing efficiencies of TALEN and CRISPR-Cas9 when targeting the human CCR5 gene

Abstract The human C-C chemokine receptor type-5 (CCR5) is the major transmembrane co-receptor that mediates HIV-1 entry into target CD4+ cells. Gene therapy to knock-out the CCR5 gene has shown encouraging results in providing a functional cure for HIV-1 infection. In gene therapy strategies, the initial region of the CCR5 gene is a hotspot for producing functional gene knock-out. Such target gene editing can be done using programmable endonucleases such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). These two gene editing approaches are the most modern and effective tools for precise gene modification. However, little is known of potential differences in the efficiencies of TALEN and CRISPR-Cas9 for editing the beginning of the CCR5 gene. To examine which of these two methods is best for gene therapy, we compared the patterns and amount of editing at the beginning of the CCR5 gene using TALEN and CRISPR-Cas9 followed by DNA sequencing. This comparison revealed that CRISPR-Cas9 mediated the sorting of cells that contained 4.8 times more gene editing than TALEN+ transfected cells.

摘要 人类C-C趋化因子受体5型（C-C chemokine receptor type-5, CCR5）是介导人类免疫缺陷病毒1型（HIV-1）侵入靶CD4阳性细胞的主要跨膜共受体。通过敲除CCR5基因的基因治疗策略，在为HIV-1感染实现功能性治愈方面展现出令人鼓舞的成果。在各类基因治疗方案中，CCR5基因的起始区域是实现功能性基因敲除的热点靶点。此类靶向基因编辑可借助可编程核酸内切酶完成，例如转录激活因子样效应物核酸酶（transcription activator-like effector nucleases, TALEN）或成簇规律间隔短回文重复序列相关蛋白9（clustered regularly interspaced short palindromic repeats-Cas9, CRISPR-Cas9），这两种基因编辑技术是当前用于精准基因修饰的最前沿且高效的工具。然而，目前针对TALEN与CRISPR-Cas9在CCR5基因起始区域的编辑效率差异，相关研究仍较为匮乏。为探究二者中更适用于基因治疗的方案，本研究采用TALEN与CRISPR-Cas9分别对CCR5基因起始区域进行编辑，并结合DNA测序对比两种方法的编辑模式与编辑效率。本次对比结果显示，CRISPR-Cas9介导的细胞基因编辑水平是转染TALEN细胞的4.8倍。