Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus
Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus
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DOI：
10.1038/s41551-023-01037-x
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发表时间：
2023-05-01
影响因子：
28.1
通讯作者：
Qi，Lei S.
中科院分区：
文献类型：
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作者：
Chavez，Michael;Rane，Draven A.;Qi，Lei S.
在原代人类细胞中定向插入和稳定表达大量基因有效载荷需要健壮、高效和易于实施的方法。通过逆转录病毒的大有效载荷插入通常是半随机的，并受到转基因沉默的阻碍。利用同源定向修复将有效载荷置于内源必需基因的控制下，可以克服沉默，但通常会导致低敲入效率和细胞毒性。在这里，我们报告了一种方法，用于敲入和稳定表达大的有效载荷，并在两个内源基因座同时敲入两个基因。这种方法，我们将其命名为CLIP(通过伪病毒进行长片段整合的CRISPR)，它利用整合酶缺陷的慢病毒编码有效载荷，在同源臂和‘切割位点’的两侧插入内源必需基因的有效载荷上游和框架内，然后通过电穿孔传递CRISPR相关核糖核蛋白复合体。我们发现，CLIP能够以较低的细胞毒性在原代T细胞中高效插入和稳定表达大有效载荷和两种难以表达的病毒抗原。CLIP为制造工程化原电池提供了一种可扩展且高效的方法。
The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for ‘CRISPR for long-fragment integration via pseudovirus’), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and ‘cut sites’ to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.