RNA 引导的人类细胞中 S 转座子的重定向。
RNA-guided retargeting of S transposition in human cells.
机构信息
Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany.
University of Applied Sciences, Giessen, Germany.
出版信息
Elife. 2020 Mar 6;9:e53868. doi: 10.7554/eLife.53868.
An ideal tool for gene therapy would enable efficient gene integration at predetermined sites in the human genome. Here we demonstrate biased genome-wide integration of the (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, and occurred in an asymmetric pattern with a bias towards sites in a relatively narrow, 300 bp window downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.
一种理想的基因治疗工具应该能够在人类基因组的预定位置实现高效的基因整合。在这里，我们通过将 SB 转座子与 CRISPR/Cas9 系统的组件结合，展示了其在全基因组范围内偏向性整合的能力。我们提供了一个概念验证，即通过将其与无催化活性的 Cas9（dCas9）融合，并提供针对人类逆转录转座子的单个向导 RNA（sgRNA），可以影响 SB 的靶位点选择。转座子整合的富集依赖于 sgRNA，并且以不对称的模式发生，偏向于 sgRNA 靶标下游相对较窄的 300bp 窗口中的位点。我们的数据表明，CRISPR/Cas9 指定的靶向机制迫使整合到基因组区域，否则这些区域是 SB 转座的不良靶标。这项技术的未来改进可能允许开发用于精确基因工程的特定基因插入方法。
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