改造RNA的二级结构可以提高CRISPR系统的特异性，这一成果由杜克大学Charles A. Gersbach团队取得。 该成果发表在2019年6月出版的国际学术期刊《Nature Biotechnology》上。

课题组研究人员表示，在单导向RNA的间隔区域(hp-sgRNAs)改造一个发夹结构的RNA二级结构，与各种CRISPR效应物结合使用，可以提高几个数量级的特异性。该团队首先证明设计hp-sgRNAs，根据化脓性链球菌来源的Cas9(SpCas9)，可以调节反式激活因子的活性。然后课题组表明hp-sgRNAs可以增加基因编辑的特异性在五个不同Cas9或Cas12a变体中。他们的结果表明RNA二级结构是一个基本参数，可以调节不同的CRISPR系统的活性。

据介绍，CRISPR (簇状规则间隔短回文重复序列)系统已经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，些细菌核酸酶出现了脱靶效应。这对于应用治疗程序的创建是潜在的危险，还可能会影响生物研究的最终结果。因此，提高这些核酸酶的精确度具有广泛的意义。

附：英文原文

Title: Increasing the specificity of CRISPR systems with engineered RNA secondary structures

Author: D. Dewran Kocak, Eric A. Josephs, Vidit Bhandarkar, Shaunak S. Adkar, Jennifer B. Kwon, Charles A. Gersbach

Issue&Volume: Volume 37 Issue 6, June 2019

Abstract: 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.

DOI: 10.1038/s41587-019-0095-1

Source:https://www.nature.com/articles/s41587-019-0095-1