Backgroundβ-Thalassemia is an inherited hematological disorder caused by mutations in the human hemoglobin beta (HBB) gene that reduce or abrogate β-globin expression. Although lentiviral-mediated expression of β-globin and autologous transplantation is a promising therapeutic approach, the risk of insertional mutagenesis or low transgene expression is apparent. However, targeted gene correction of HBB mutations with programmable nucleases such as CRISPR/Cas9, TALENs, and ZFNs with non-viral repair templates ensures a higher safety profile and endogenous expression control.MethodsWe have compared three different gene-editing tools (CRISPR/Cas9, TALENs, and ZFNs) for their targeting efficiency of the HBB gene locus. As a proof of concept, we studied the personalized gene-correction therapy for a common β-thalassemia splicing variant HBBIVS1–110 using Cas9 mRNA and several optimally designed single-stranded oligonucleotide (ssODN) donors in K562 and CD34+ hematopoietic stem cells (HSCs).ResultsOur results exhibited that indel frequency of CRISPR/Cas9 was superior to TALENs and ZFNs (P < 0.0001). Our designed sgRNA targeting the site of HBBIVS1–110 mutation showed indels in both K562 cells (up to 77%) and CD34+ hematopoietic stem cells—HSCs (up to 87%). The absolute quantification by next-generation sequencing showed that up to 8% site-specific insertion of the NheI tag was achieved using Cas9 mRNA and a chemically modified ssODN in CD34+ HSCs.ConclusionOur approach provides guidance on non-viral gene correction in CD34+ HSCs using Cas9 mRNA and chemically modified ssODN. However, further optimization is needed to increase the homology directed repair (HDR) to attain a real clinical benefit for β-thalassemia.

中文翻译：

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使用 Cas9 mRNA 和 ssODN 供体对 CD34+ 造血干细胞中的 HBB 突变进行基因校正

背景β-地中海贫血是一种遗传性血液病，由人类血红蛋白β (HBB) 基因突变导致β-珠蛋白表达降低或消失。尽管慢病毒介导的 β-珠蛋白表达和自体移植是一种很有前景的治疗方法，但插入突变或低转基因表达的风险是显而易见的。然而，使用具有非病毒修复模板的 CRISPR/Cas9、TALEN 和 ZFNs 等可编程核酸酶对 HBB 突变进行靶向基因校正可确保更高的安全性和内源性表达控制。方法我们比较了三种不同的基因编辑工具（CRISPR/Cas9 、TALEN 和 ZFN) 的 HBB 基因位点靶向效率。作为概念证明，我们使用 Cas9 mRNA 和几个优化设计的单链寡核苷酸 (ssODN) 供体在 K562 和 CD34+ 造血干细胞 (HSC) 中研究了常见 β-地中海贫血剪接变体 HBBIVS1-110 的个性化基因校正疗法。结果我们的结果表明插入CRISPR/Cas9 的频率优于 TALENs 和 ZFNs（P < 0.0001）。我们设计的靶向 HBBIVS1-110 突变位点的 sgRNA 在 K562 细胞（高达 77%）和 CD34+ 造血干细胞 - HSC（高达 87%）中均显示插入缺失。下一代测序的绝对定量表明，使用 Cas9 mRNA 和化学修饰的 ssODN 在 CD34+ HSCs 中实现了高达 8% 的 NheI 标签位点特异性插入。结论我们的方法为 CD34+ HSCs 中的非病毒基因校正提供了指导Cas9 mRNA 和化学修饰的 ssODN。然而，