Rapid and Simple Screening of CRISPR Guide RNAs (gRNAs) in Cultured Cells Using Adeno-Associated Viral (MV) Vectors
Rapid and Simple Screening of CRISPR Guide RNAs (gRNAs) in Cultured Cells Using Adeno-Associated Viral (MV) Vectors
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DOI：
10.1007/978-1-4939-9170-9_8
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发表时间：
2019-01-01
期刊：
影响因子：
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通讯作者：
Grimm, Dirk
中科院分区：
文献类型：
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作者：
Fakhiri, Julia;Nickl, Manuela;Grimm, Dirk
包括最近推出的 CRISPR/Cas9 系统在内的基因组编辑试剂已成为成熟并广泛使用的分子工具，用于回答基本的生物学问题以及靶向和治疗遗传疾病。最初源自细菌和古细菌的 CRISPR 系统可以使用不同的技术递送到细胞中，包括 (1) mRNA 或质粒 DNA 的转染，(2) 电穿孔质粒 DNA 或 Cas9 蛋白与 g(uide)RNA 的复合物，或 (3) 使用非病毒或病毒载体。在后者中，腺相关病毒 (AAV) 由于许多有利的特性而特别有吸引力：(1) 其无致病性和附加型持久性，(2) 病毒生产和纯化的简便性，(3) 在最低生物安全 1 级条件下的安全处理，以及 (4) 具有多种天然血清型和具有不同细胞特异性的合成衣壳变体的可用性。在这里，我们描述了一种快速、简单的方案，用于将 CRISPR/Cas9 组件小规模包装到 AAV 载体中。为了展示其潜力，我们采用这种方法筛选针对 Hepal-6 细胞中的小鼠 miR-122 位点（使用 AAV 血清型 6，AAV6）或 HeLaP4-NLtr 细胞中的人类免疫缺陷病毒 (HIV) 5'LTR（使用合成的 AAV9 变体）的 gRNA。我们还提供了用于大规模生产纯化 AAV/CRISPR 载体库存的详细方案，该方案允许更高的体外切割效率，并且适合直接体内应用。
Genome editing reagents including the recently introduced CRISPR/Cas9 system have become established and widely used molecular tools to answer fundamental biological questions and to target and treat genetic diseases. The CRISPR system, originally derived from bacteria and archaea, can be delivered into cells using different techniques, comprising (1) transfection of mRNA or plasmid DNA, (2) electroporation of plasmid DNA or the Cas9 protein in a complex with a g(uide)RNA, or (3) use of nonviral or viral vectors. Among the latter, Adeno-associated viruses (AAVs) are particularly attractive owing to many favorable traits: (1) their apathogenicity and episomal persistence, (2) the ease of virus production and purification, (3) the safe handling under lowest biosafety level 1 conditions, and (4) the availability of numerous natural serotypes and synthetic capsid variants with distinct cell specificities. Here, we describe a fast and simple protocol for small-scale packaging of CRISPR/Cas9 components into AAV vectors. To showcase its potential, we employ this method for screening of gRNAs targeting the murine miR-122 locus in Hepal-6 cells (using AAV serotype 6, AAV6) or the 5'LTR of the human immunodeficiency virus (HIV) in HeLaP4-NLtr cells (using a synthetic AAV9 variant). We furthermore provide a detailed protocol for large-scale production of purified AAV/CRISPR vector stocks that permit higher cleavage efficiencies in vitro and are suitable for direct in vivo applications.