The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer–target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects.

精确 RNA 编辑工具的开发对于 RNA 治疗学的发展至关重要。CRISPR（成簇的规则间隔短回文重复序列）PspCas13b 是一种可编程的 RNA 核酸酶，由于其 30 个核苷酸的间隔序列而被预测具有卓越的特异性。然而，其设计原则及其目标、脱靶和附带活动仍然没有得到很好的描述。在这里，我们提出了单碱基平铺筛选和计算分析，以确定人类细胞中有效和高选择性 RNA 识别和切割的关键设计原则。我们表明，在精确位置含有鸟苷碱基的间隔区的从头设计可以大大提高低效 CRISPR RNA （crRNA） 的催化活性。这些经过验证的设计原则（集成到在线工具 https://cas13target.azurewebsites.net/） 中）可以预测高效 crRNA，准确率为 ~90%。此外，全面的间隔区-靶标诱变显示，PspCas13b 只能容忍多达 4 个错配，并且需要与靶标配对 ~26 个核苷酸的碱基配对才能激活其核酸酶结构域，突出了与其他 RNA 或 DNA 干扰工具相比的优异特异性。基于这种靶向分辨率，我们预测 PspCas13b 对其他细胞转录物具有脱靶效应的可能性极低。蛋白质组学分析验证了这一预测，并表明与其他 Cas13 直系同源物不同，PspCas13b 表现出有效的靶向活性并且缺乏附带效应。