Increasing the specificity of CRISPR systems with engineered RNA secondary structures
Increasing the specificity of CRISPR systems with engineered RNA secondary structures
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DOI：
10.1038/s41587-019-0095-1
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发表时间：
2019-06-01
影响因子：
46.9
通讯作者：
Gersbach, Charles A.
中科院分区：
文献类型：
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作者：
Kocak, D. Dewran;Josephs, Eric A.;Gersbach, Charles A.
CRISPR（聚集规则间隔短回文重复序列）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。这对治疗应用产生了潜在的危害，并可能混淆生物学研究的结果。因此，提高这些核酸酶的精度具有广泛的意义。本研究表明，在单导rna （hp-sgRNAs）的间隔区域上设计一个发夹二级结构，当与各种CRISPR效应物结合时，可以将特异性提高几个数量级。我们首先证明了设计的hp-sgRNAs可以调节基于化脓性链球菌Cas9 （SpCas9）的反激活子的活性。然后，我们证明hp- sgrna使用五种不同的Cas9或Cas12a变体增加了基因编辑的特异性。我们的研究结果表明，RNA二级结构是调节多种CRISPR系统活性的基本参数。
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.