Department of Biomedical Engineering, Duke University, Durham, NC, USA.
Center for Genomic and Computational Biology, Duke University, Durham, NC, USA.
Nat Biotechnol. 2019 Jun;37(6):657-666. doi: 10.1038/s41587-019-0095-1. Epub 2019 Apr 15.
CRISPR (clustered regularly interspaced short palindromic repeat) systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. In some cases, these bacterial nucleases have demonstrated off-target activity. This creates a potential hazard for therapeutic applications and could confound results in biological research. Therefore, improving the precision of these nucleases is of broad interest. Here we show that engineering a hairpin secondary structure onto the spacer region of single guide RNAs (hp-sgRNAs) can increase specificity by several orders of magnitude when combined with various CRISPR effectors. We first demonstrate that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from Streptococcus pyogenes (SpCas9). We then show that hp-sgRNAs increase the specificity of gene editing using five different Cas9 or Cas12a variants. Our results demonstrate that RNA secondary structure is a fundamental parameter that can tune the activity of diverse CRISPR systems.
CRISPR（成簇规律间隔短回文重复）系统已广泛应用于基础科学、生物技术、基因和细胞治疗。在某些情况下，这些细菌核酸酶表现出脱靶活性。这给治疗应用带来了潜在的危险，并可能混淆生物研究的结果。因此，提高这些核酸酶的精度是人们广泛关注的问题。在这里，我们展示了在单引导 RNA（sgRNA）的间隔区构建发夹二级结构（hp-sgRNA）可以与各种 CRISPR 效应物结合，将特异性提高几个数量级。我们首先证明，设计的 hp-sgRNA 可以调节基于化脓性链球菌 Cas9 的转录激活因子（SpCas9）的活性。然后我们表明，hp-sgRNA 提高了使用五种不同 Cas9 或 Cas12a 变体进行基因编辑的特异性。我们的结果表明，RNA 二级结构是一个可以调节不同 CRISPR 系统活性的基本参数。
