The Cas9/guide RNA (Cas9/gRNA) system is commonly used for genome editing. mRNA expressing Cas9 can induce innate immune responses, reducing Cas9 expression. First-generation Cas9 mRNAs were modified with pseudouridine and 5-methylcytosine to reduce innate immune responses. We combined four approaches to produce more active, less immunogenic second-generation Cas9 mRNAs. First, we developed a novel co-transcriptional capping method yielding natural Cap 1. Second, we screened modified nucleotides in Cas9 mRNA to identify novel modifications that increase Cas9 activity. Third, we depleted the mRNA of uridines to improve mRNA activity. Lastly, we tested high-performance liquid chromatography (HPLC) purification to remove double-stranded RNAs. The activity of these mRNAs was tested in cell lines and primary human CD34+ cells. Cytokines were measured in whole blood and mice. These approaches yielded more active and less immunogenic mRNA. Uridine depletion (UD) most impacted insertion or deletion (indel) activity. Specifically, 5-methoxyuridine UD induced indel frequencies as high as 88% (average ± SD = 79% ± 11%) and elicited minimal immune responses without needing HPLC purification. Our work suggests that uridine-depleted Cas9 mRNA modified with 5-methoxyuridine (without HPLC purification) or pseudouridine may be optimal for the broad use of Cas9 both in vitro and in vivo.

Cas9 /指南RNA（Cas9 / gRNA）系统通常用于基因组编辑。表达Cas9的mRNA可以诱导先天性免疫反应，从而降低Cas9的表达。用伪尿苷和5-甲基胞嘧啶修饰第一代Cas9 mRNA，以减少先天免疫应答。我们结合了四种方法来产生更活跃，免疫原性较低的第二代Cas9 mRNA。首先，我们开发了一种可产生天然Cap 1的新型共转录加帽方法。其次，我们在Cas9中筛选了修饰的核苷酸mRNA鉴定增加Cas9活性的新修饰。第三，我们耗尽了尿苷的mRNA以提高mRNA活性。最后，我们测试了高效液相色谱（HPLC）纯化以去除双链RNA。在细胞系和原代人CD34 +细胞中测试了这些mRNA的活性。在全血和小鼠中测量细胞因子。这些方法产生了更多的活性和更少的免疫原性mRNA。尿苷耗竭（UD）对插入或缺失（indel）活性的影响最大。具体而言，5-甲氧基尿苷UD诱导的插入缺失频率高达88％（平均±SD = 79％±11％），并且不需要HPLC纯化即可引起最小的免疫反应。我们的工作表明尿苷耗尽的Cas9mRNA的修饰的5-methoxyuridine（无HPLC纯化）或假尿苷可能是最佳的同时为广泛使用的Cas9在体外和体内。