The RNA-guided, modified type II prokaryotic clustered regularly interspaced palindromic repeats with CRISPR-associated proteins (CRISPR/Cas9) system represents a simple gene editing platform with applications in biotechnology and also potentially as a therapeutic modality. The system requires a small guide RNA (sgRNA) and a catalytic Cas9 protein to induced non-homologous end joining (NHEJ) at break sites resulting in the formation of inactivating mutations, or through homology-directed repair (HDR) can engineer in specific sequence changes. Although CRISPR/Cas9 is a powerful technology, the effects can be limited as a result of nuclease-mediated degradation of the RNA components. Significant research has focused on the solid-phase synthesis of CRISPR RNA components with chemically modified bases, but this approach is technically challenging and expensive. Development of a simple, generic approach to generate chemically modified CRISPR RNAs may broaden applications that require nuclease-resistant CRISPR components. We report here the development of a novel, functional U-replaced tracrRNA that can be in vitro transcribed with chemically stabilizing 2'F-pyrimidines. These data represent a unique and facile approach to generating chemically stabilized CRISPR RNA.

RNA引导、修饰的II型原核生物簇状规则间隔回文重复序列与CRISPR相关蛋白（CRISPR/Cas9）系统代表了一个简单的基因编辑平台，可在生物技术中应用，也有可能作为一种治疗方式。该系统需要小引导RNA (sgRNA) 和催化Cas9 蛋白在断裂位点诱导非同源末端连接(NHEJ)，从而形成失活突变，或者通过同源定向修复(HDR) 可以设计特定序列变化。尽管 CRISPR/Cas9 是一项强大的技术，但由于核酸酶介导的 RNA 成分降解，其效果可能会受到限制。重要的研究集中在具有化学修饰碱基的 CRISPR RNA 成分的固相合成上，但这种方法在技术上具有挑战性且昂贵。开发一种简单、通用的方法来生成化学修饰的 CRISPR RNA 可能会拓宽需要耐核酸酶 CRISPR 组件的应用。我们在此报告了一种新型功能性 U 取代 tracrRNA 的开发，该 tracrRNA 可以用化学稳定的 2'F-嘧啶进行体外转录。这些数据代表了一种独特且简便的生成化学稳定 CRISPR RNA 的方法。