Off-target effects of CRISPRa on interleukin-6 expression

Inactive fusion variants of the CRISPR-Cas9 system are increasingly being used as standard methodology to study transcription regulation. Their ability to readily manipulate the native genomic loci is particularly advantageous. In this work, we serendipitously uncover the key cytokine IL6 as an off-target of the activating derivative of CRISPR (CRISPRa) while studying RP11-326A19.4, a novel long-non coding RNA (lncRNA). Increasing RP11-326A19.4 expression in HEK293T cells via CRISPRa-mediated activation of its promoter region induced genome-wide transcriptional changes, including upregulation of IL6, an important cytokine. IL6 was increased in response to distinct sgRNA targeting the RP11-326A19.4 promoter region, suggesting specificity. Loss of the cognate sgRNA recognition sites failed to abolish CRISPRa mediated activation of IL6 however, pointing to off-target effects. Bioinformatic approaches did not reveal predicted off-target binding sites. Off-target activation of IL6 was sustained and involved low level activation of known IL6 regulators. Increased IL6 remained sensitive to further activation by TNFα, consistent with the existence of independent mechanisms. This study provides experimental evidence that CRISPRa has discrete, unpredictable off-targeting limitations that must be considered when using this emerging technology.

失活融合型CRISPR-Cas9系统变体正日益成为转录调控研究的标准化实验方法。其能够便捷操控内源基因组位点的特性尤为突出。本研究在研究新型长链非编码RNA（long non-coding RNA, lncRNA）RP11-326A19.4时，意外发现关键细胞因子白介素6（IL6）是CRISPR激活系统（CRISPRa）的脱靶效应靶点。通过CRISPRa介导激活HEK293T细胞中RP11-326A19.4的启动子区域以提升其表达，可诱导全基因组转录改变，其中包括重要细胞因子IL6的上调。针对RP11-326A19.4启动子区域的不同单导RNA（single guide RNA, sgRNA）均可引起IL6水平升高，这提示该现象具有特异性。然而，敲除同源sgRNA识别位点并不能阻断CRISPRa介导的IL6激活，这表明存在脱靶效应。生物信息学分析未发现预测的脱靶结合位点。IL6的脱靶激活持续存在，且伴随已知IL6调控因子的低水平激活。升高的IL6仍可被肿瘤坏死因子α（tumor necrosis factor α, TNFα）进一步激活，这与独立调控机制的存在相符。本研究提供实验证据表明，CRISPRa存在离散且不可预测的脱靶局限性，在使用这一新兴技术时必须加以考量。