Expanding the Potential of Mammalian Genome Engineering via Targeted DNA Integration,ACS Synthetic Biology

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Expanding the Potential of Mammalian Genome Engineering via Targeted DNA Integration
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-02-17 , DOI: 10.1021/acssynbio.0c00576
Meng Zhang _{1,

2} , Che Yang _{2,

3} , Ipek Tasan _{2,

3} , Huimin Zhao _{1,

2,

3,

4,

5}

Affiliation

Inserting custom designed DNA sequences into the mammalian genome plays an essential role in synthetic biology. In particular, the ability to introduce foreign DNA in a site-specific manner offers numerous advantages over random DNA integration. In this review, we focus on two mechanistically distinct systems that have been widely adopted for targeted DNA insertion in mammalian cells, the CRISPR/Cas9 system and site-specific recombinases. The CRISPR/Cas9 system has revolutionized the genome engineering field thanks to its high programmability and ease of use. However, due to its dependence on linearized DNA donor and endogenous cellular pathways to repair the induced double-strand break, CRISPR/Cas9-mediated DNA insertion still faces limitations such as small insert size, and undesired editing outcomes via error-prone repair pathways. In contrast, site-specific recombinases, in particular the Serine integrases, demonstrate large-cargo capability and no dependence on cellular repair pathways for DNA integration. Here we first describe recent advances in improving the overall efficacy of CRISPR/Cas9-based methods for DNA insertion. Moreover, we highlight the advantages of site-specific recombinases over CRISPR/Cas9 in the context of targeted DNA integration, with a special focus on the recent development of programmable recombinases. We conclude by discussing the importance of protein engineering to further expand the current toolkit for targeted DNA insertion in mammalian cells.

中文翻译：

通过靶向 DNA 整合扩大哺乳动物基因组工程的潜力

将定制设计的 DNA 序列插入哺乳动物基因组中在合成生物学中发挥着重要作用。特别是，以位点特异性方式引入外源 DNA 的能力比随机 DNA 整合具有许多优势。在这篇综述中，我们重点关注两种机制不同的系统，即 CRISPR/Cas9 系统和位点特异性重组酶，这两种系统已广泛用于哺乳动物细胞中的靶向 DNA 插入。 CRISPR/Cas9系统凭借其高度可编程性和易用性彻底改变了基因组工程领域。然而，由于其依赖线性化 DNA 供体和内源细胞途径来修复诱导的双链断裂，CRISPR/Cas9 介导的 DNA 插入仍然面临局限性，例如插入尺寸小，以及通过容易出错的修复途径产生不期望的编辑结果。相比之下，位点特异性重组酶，特别是丝氨酸整合酶，表现出大货物能力并且不依赖于 DNA 整合的细胞修复途径。在这里，我们首先描述了在提高基于 CRISPR/Cas9 的 DNA 插入方法的整体功效方面的最新进展。此外，我们强调了在靶向 DNA 整合方面，位点特异性重组酶相对于 CRISPR/Cas9 的优势，特别关注可编程重组酶的最新发展。最后，我们讨论了蛋白质工程对于进一步扩展当前在哺乳动物细胞中定向 DNA 插入的工具包的重要性。

更新日期：2021-03-19

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