An optimized design for single copy short hairpin RNAi

 描述（由申请人提供）：短发夹RNA（shRNA）介导的RNAi是研究癌症途径的成熟方法，是一种有前途的未来治疗策略。特别地，通过逆转录病毒转导递送shRNA能够在小鼠模型和难以转染的细胞系中实现RNAi敲低。基于逆转录病毒shRNA的实验的主要缺点是当整合单个拷贝的shRNA载体时经常观察到的靶基因敲减减少。我们使用我们以前开发的一种新的深度测序策略研究了shRNA前体的命运。我们发现超过99%的前体分子被转移到非生产性的生物合成途径，严重限制了成熟RNA的产量。在这个提议的应用中，我们描述了一种筛选策略，以优化shRNA设计并最大化靶基因敲低潜力。我们的长期目标，超出了这个拨款申请的范围，是使用我们优化的shRNA设计构建全基因组的shRNA文库。

 DESCRIPTION (provided by applicant): Short hairpin RNA (shRNA) mediated RNAi is a well-established method for investigating cancer pathways and is a promising future therapeutic strategy. In particular, delivery of shRNAs by retroviral transduction enables RNAi knockdown in mouse models and cell lines refractory to transfection. The primary disadvantage of retroviral shRNA-based experiments is the reduced target gene knockdown frequently observed when a single copy of the shRNA vector is integrated. We have investigated the fate of shRNA precursors using a novel deep sequencing strategy we have previously developed. We find over 99 percent of precursor molecules are diverted to a non-productive biogenesis route, severely limiting the amount of mature RNA produced. In this proposed application we describe a screening strategy to optimize shRNA design and maximize target gene knockdown potential. Our long term goal, beyond the scope of this grant application, is to construct genome-wide shRNA libraries using our optimized shRNA design.