Here, we report that CRISPR guide RNAs (gRNAs) with a 5′-triphosphate group (5′-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5′-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5′-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4⁺ T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5′-hydroxyl group are much more efficient than in vitro–transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.

在这里，我们报道通过体外转录产生的具有5'-三磷酸基团（5'-ppp gRNA）的CRISPR指导RNA（gRNA）触发人和鼠细胞中RNA感知的先天免疫应答，从而导致细胞毒性。DDX58识别胞浆中的5'-ppp gRNA，进而激活I型干扰素反应，导致约80％的细胞死亡。我们显示三磷酸基团可以在体外被磷酸酶去除，并且与Cas9或Cpf1形成复合的5'-羟基gRNA避免了先天免疫反应，并且可以在主要人类CD4 ^+中以95％的频率实现定向诱变T细胞。这些结果与以前的发现相符，即在人和其他哺乳动物细胞中，化学合成的具有5'-羟基的sgRNA比体外转录（IVT）sgRNA效率更高。IVT sgRNA的磷酸酶处理是制备高活性sgRNA的一种经济高效的方法，可避免人细胞中的先天免疫应答。