Developing oncolytic Herpes simplex virus type 1 through UL39 knockout by CRISPR-Cas9
Developing oncolytic Herpes simplex virus type 1 through UL39 knockout by CRISPR-Cas9
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DOI：
10.22038/ijbms.2020.43864.10286
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发表时间：
2020-07-01
影响因子：
2.2
通讯作者：
Teimoori, Ali
中科院分区：
文献类型：
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作者：
Ebrahimi, Saeedeh;Makvandi, Manoochehr;Teimoori, Ali
目标：溶瘤单纯疱疹病毒 1 型 (HSV-1) 已成为一种有前途的癌症治疗策略。然而，由于传统基因组编辑方法的效率低下，新型溶瘤突变体的开发仍然是一个重大挑战。最近，CRISPR-Cas9 彻底改变了基因组编辑。 材料和方法：在本研究中，我们旨在评估 CRISPR-Cas9 操纵 UL39 基因产生溶瘤 HSV-1 的能力。本文针对 UL39 基因设计了 3 个 sgRNA，将其转染至 HEK-293 细胞系中，然后用 HSV-1 KOS 进行感染。结果：经过三轮空斑纯化，通过 PCR 分析和测序鉴定出几种 HSV-1 突变体。其中一个删除了 55 个核苷酸的突变导致了移码突变，进而产生了一种截短的蛋白质，其中 1137 个氨基酸中只有 167 个氨基酸。 Vero 和原代成纤维细胞的功能分析表明，与 HSV-1 KOS 相比，HSV-1 突变体的病毒复制显着降低，噬斑尺寸更小。此外，与 HSV-1 KOS 相比，HSV-1 突变体感染细胞（Vero 细胞和原代成纤维细胞）上清液中病毒基因组的相对数量显着减少。结论：我们的数据表明，用 CRISPR-Cas9 靶向 UL39 可以形成溶瘤 HSV-1。
Objective(s): Oncolytic Herpes simplex virus type 1 (HSV-1) has emerged as a promising strategy for cancer therapy. However, development of novel oncolytic mutants has remained a major challenge owing to low efficiency of conventional genome editing methods. Recently, CRISPR-Cas9 has revolutionized genome editing.Materials and Methods: In this study, we aimed to evaluate the capability of CRISPR-Cas9 to manipulate the UL39 gene to create oncolytic HSV-1. Herein, three sgRNAs were designed against the UL39 gene and transfected into HEK-293 cell line followed by infection with HSV-1 KOS.Results: After three rounds of plaque purification, several HSV-1 mutants were identified by PCR analysis and sequencing. One of these mutations in which 55 nucleotides were deleted resulted in a frameshift mutation that in turn produced a truncated protein with only 167 amino acids from 1137 amino acids. Functional analysis in Vero and primary fibroblast cells revealed that viral replication was significantly lower and plaque size was smaller in the HSV-1 mutant compared with HSV-1 KOS. Moreover, the relative amount of viral genome present in the supernatants of infected cells (Vero and primary fibroblast cells) with HSV-1 mutant was significantly decreased compared with those of HSV-1 KOS.Conclusion: Our data revealed that targeting UL39 with CRISPR-Cas9 could develop oncolytic HSV-1.