Scaffold-mediated non-viral delivery platform for CRISPR/Cas9-based genome editing
Scaffold-mediated non-viral delivery platform for CRISPR/Cas9-based genome editing
复制标题
DOI：
10.1016/j.actbio.2019.04.020
复制
发表时间：
2019-05-01
影响因子：
9.7
通讯作者：
Chew, Sing Yian
中科院分区：
文献类型：
--
作者：
Chin, Jiah Shin;Chooi, Wai Hon;Chew, Sing Yian
基因组编辑，特别是通过简单而通用的II型CRISPR/Cas9系统，提供了一种精确控制细胞命运的有效途径，这是组织再生的一个重要方面。不幸的是，大多数CRISPR/Cas9非病毒递送策略仅利用微米/纳米颗粒递送方法。虽然这些方法提供了合理的基因组编辑效率，但它们的全身递送可能导致不期望的脱靶效应。对于体内应用，更局部化和持续的递送方法可能是有用的，特别是在组织再生的情况下。在这里，我们开发了一种支架，以局部和非病毒的方式递送CRISPR/Cas9组分（即单向导RNA（sgRNA）和Cas9蛋白复合物）。具体地，使用贻贝启发的生物粘附涂层，聚DOPA-黑色素（pDOPA），我们将Cas9：sgRNA脂质体复合物吸附到生物模拟纤维支架上。为了评估该平台的基因组编辑效率，使用U2OS.EGFP细胞作为模型细胞类型。pDOPA包被对于允许Cas9：sgRNA脂质体转染胺复合物以更高的装载效率粘附到支架上是必不可少的，而层粘连蛋白包被对于维持pDOPA包被的纤维上的细胞活力和增殖以进行有效的基因编辑是必要的（21.5%编辑效率，p < 0.001）。重要的是，U2OS.EGFP细胞通过反向转染直接从支架摄取Cas9：sgRNA脂质体复合物。总之，我们证明了这种纤维支架在提供Cas9：sgRNA复合物的局部、持续和非病毒递送中的功效。这种基因组编辑支架可能在组织再生中找到有用的应用。重要性声明目前，缺乏有效的非病毒手段来递送CRISPR/Cas9组分用于基因组编辑。大多数现有方法仅通过注射或全身递送利用微米/纳米颗粒，这可能导致不期望的脱靶效应。在这里，我们报告了一个以局部和持续方式递送CRISPR/Cas9组分（即单向导RNA（sgRNA）和Cas9蛋白复合物）的平台。我们使用贻贝启发的生物粘附涂层来用Cas9：sgRNA脂质体复合物功能化生物模拟纤维支架，以允许对接种在支架上的细胞进行有效的基因编辑。重要的是，细胞直接从支架上摄取Cas9：sgRNA脂质体复合物。这种基因组编辑支架可能在组织再生中找到有用的应用。(C)2019 Acta Materialia Inc.由爱思唯尔有限公司出版。保留所有权利。
Genome editing, especially via the simple and versatile type II CRISPR/Cas9 system, offers an effective avenue to precisely control cell fate, an important aspect of tissue regeneration. Unfortunately, most CRISPR/Cas9 non-viral delivery strategies only utilise micro-/nano-particle delivery methods. While these approaches provide reasonable genomic editing efficiencies, their systemic delivery may lead to undesirable off-target effects. For in vivo applications, a more localized and sustained delivery approach may be useful, particularly in the context of tissue regeneration. Here, we developed a scaffold that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and non-viral manner. Specifically, using mussel-inspired bioadhesive coating, polyDOPA-melanin (pDOPA), we adsorbed Cas9:sgRNA lipofectamine complexes onto bio-mimicking fiber scaffolds. To evaluate the genome-editing efficiency of this platform, U2OS.EGFP cells were used as the model cell type. pDOPA coating was essential in allowing Cas9:sgRNA lipofectamine complexes to adhere onto the scaffolds with a higher loading efficiency, while laminin coating was necessary for maintaining cell viability and proliferation on the pDOPA-coated fibers for effective gene editing (21.5% editing efficiency, p < 0.001). Importantly, U2OS.EGFP cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds via reverse transfection. Overall, we demonstrate the efficacy of such fiber scaffolds in providing localized, sustained and non-viral delivery of Cas9:sgRNA complexes. Such genome editing scaffolds may find useful applications in tissue regeneration.Statement of significanceCurrently, there is a lack of effective non-viral means to deliver CRISPR/Cas9 components for genome editing. Most existing approaches only utilize micro-/nano-particles by injection or systemic delivery, which may lead to undesirable off-target effects. Here, we report a platform that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and sustained manner. We used mussel-inspired bioadhesive coating to functionalize the bio-mimicking fiber scaffolds with Cas9:sgRNA lipofectamine complexes, to allow effective gene editing for the cells seeded on the scaffolds. Importantly, the cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds. Such genome editing scaffolds may find useful applications in tissue regeneration. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.