CRISPR/Cas9-mediated transcriptional activation (CRISPRa) is a powerful tool for investigating complex biological phenomena. Although CRISPRa approaches based on VP64 have been widely studied in both cultured cells and in animal models and exhibit great versatility for various cell types and developmental stages in vivo, different dCas9-VP64 versions have not been rigorously compared. Here, we compared different dCas9-VP64 constructs in identical contexts, including the cell lines used and the transfection conditions, for their ability to activate endogenous and exogenous genes. Moreover, we investigated the optimal approach for VP64 addition to VP64- and p300-based constructs. We found that MS2-MCP-scaffolded VP64 enhanced dCas9-VP64 and dCas9-p300 activity better than did direct VP64 fusion to the N-terminus of dCas9. dCas9-VP64+MCP-VP64 and dCas9-p300+MCP-VP64 were superior to VP64-dCas9-VP64 for all target genes tested. Furthermore, multiplexing gRNA expression with dCas9-VP64+MCP-VP64 or dCas9-p300+MCP-VP64 significantly enhanced endogenous gene activation to a level comparable to CRISPRa-SAM with a single gRNA. Our findings demonstrate improvement of the dCas9-VP64 CRISPRa system and contribute to development of a versatile, efficient CRISPRa platform.

中文翻译：

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dCas9-VP64变体用于CRISPR激活的比较分析和合理设计

CRISPR/Cas9 介导的转录激活 (CRISPRa) 是研究复杂生物现象的强大工具。虽然CRISPRa方法的基础上VP64已被广泛研究在这两个培养的细胞和动物模型和各种细胞类型和发育阶段表现出强大的多功能性在体内，不同的dCas9-VP64版本没有严格比较。在这里，我们比较了相同背景下不同 dCas9-VP64 构建体，包括使用的细胞系和转染条件，以了解它们激活内源和外源基因的能力。此外，我们研究了将 VP64 添加到基于 VP64 和 p300 的构建体的最佳方法。我们发现 MS2-MCP 支架的 VP64 增强了 dCas9-VP64 和 dCas9-p300 的活性，而不是直接将 VP64 融合到 dCas9 的 N 端。对于所有测试的靶基因，dCas9-VP64+MCP-VP64 和 dCas9-p300+MCP-VP64 均优于 VP64-dCas9-VP64。此外，dCas9-VP64+MCP-VP64 或 dCas9-p300+MCP-VP64 的多重 gRNA 表达显着增强了内源性基因激活，其水平可与具有单个 gRNA 的 CRISPra-SAM 相媲美。