The CRISPR-Cas9 system has been adapted for transcriptional activation (CRISPRa) and several second-generation CRISPRa systems (including VPR, SunTag, and SAM) have been developed to recruit different transcriptional activators to a deactivated Cas9, which is guided to a transcriptional start site via base complementarity with a target guide RNA. Multiple studies have shown the benefit of CRISPRa using plasmid or lentiviral expressed guide RNA, but the use of synthetic guide RNA has not been reported. Here we demonstrate the effective use of synthetic guide RNA for gene activation via CRISPRa. CRISPRa crRNA may be used with a canonical tracrRNA using the VPR or SunTag activation systems or with an extended tracrRNA containing an aptamer sequence for the SAM system. Transcriptional activation with synthetic crRNA:tracrRNA is comparable to activation achieved with expression vectors and combining several crRNA sequences targeting the same gene can enhance transcriptional activation. The use of synthetic crRNA is also ideal for simultaneous activation of multiple genes or use with dCas9-VPR mRNA when viral transduction is not feasible. Here, we perform a proof-of-principle arrayed screen using a CRISPRa crRNA library consisting of 153 cytokine receptor targets to identify regulators of IL-6 cytokine secretion. Together, these results demonstrate the suitability of synthetic CRISPRa guide RNA for high throughput, arrayed screening applications which allow for more complex phenotypic readouts to complement viability and drug resistance assays typically used in a pooled screening format.

CRISPR-Cas9系统已针对转录激活（CRISPRa）进行了改造，并且已经开发了多个第二代CRISPRa系统（包括VPR，SunTag和SAM），以将不同的转录激活因子募集到已失活的Cas9中，从而指导转录开始通过与靶标指导RNA的碱基互补性定位。多项研究表明，使用质粒或慢病毒表达的指导RNA可使CRISPRa受益，但尚未报道合成指导RNA的使用。在这里，我们证明了合成指导RNA通过CRISPRa激活基因的有效利用。CRISPRa crRNA可以与使用VPR或SunTag激活系统的规范tracrRNA一起使用，或与包含SAM系统的适体序列的扩展tracrRNA一起使用。合成crRNA的转录激活：tracrRNA可与表达载体实现的激活相媲美，结合靶向同一基因的多个crRNA序列可增强转录激活。当病毒转导不可行时，合成crRNA也可用于同时激活多个基因或与dCas9-VPR mRNA一起使用。在这里，我们使用由153个细胞因子受体靶组成的CRISPRa crRNA文库进行原理证明的阵列筛选，以鉴定IL-6细胞因子分泌的调节剂。总之，这些结果证明了合成CRISPRa指导RNA在高通量，阵列筛选应用中的适用性，从而允许更复杂的表型读数，以补充通常以合并筛选形式使用的活力和耐药性分析。