Cas13是迄今为止发现的唯一CRISPR/Cas系统,在保持染色体完整性的同时靶向RNA链。Cas13b或Cas13d通过crRNA指导切割RNA。然而,间隔序列特征的影响,例如长度和序列偏好,Cas13b和Cas13d的活性尚不清楚。我们的研究表明Cas13b和Cas13d对gRNA的序列组成都没有特别的偏好,包括crRNA的序列及其在靶RNA上的侧翼位点。然而,crRNA,与目标RNA的中间部分互补,似乎对Cas13b和Cas13d都显示出更高的切割效率。至于crRNA的长度,Cas13b最合适的crRNA长度是22-25nt,短至15nt的crRNA仍然是有功能的。然而,Cas13d需要更长的crRNA,和22-30ntcrRNA可以达到良好的效果。Cas13b和Cas13d均显示出加工前体crRNA的能力。我们的研究表明,Cas13b可能比Cas13d具有更强的前体加工能力。关于Cas13b或Cas13d在哺乳动物中的应用的体内研究很少。用转基因小鼠和尾静脉流体动力学注射的方法,我们的研究表明,它们在体内对靶RNA都有很高的敲低效率。这些结果表明,Cas13b和Cas13d在体内RNA操作和疾病治疗方面具有巨大潜力,而不会破坏基因组DNA。本文受版权保护。保留所有权利。
Cas13 are the only CRISPR/Cas systems found so far, which target RNA strand while preserving chromosomal integrity. Cas13b or Cas13d cleaves RNA by the crRNA guidance. However, the effect of the characteristics of the spacer sequences, such as the length and sequence preference, on the activity of Cas13b and Cas13d remains unclear. Our study shows that neither Cas13b nor Cas13d has a particular preference for the sequence composition of gRNA, including the sequence of crRNA and its flanking sites on target RNA. However, the crRNA, complementary to the middle part of the target RNA, seems to show higher cleavage efficiency for both Cas13b and Cas13d. As for the length of crRNAs, the most appropriate crRNA length for Cas13b is 22-25 nt and crRNA as short as 15 nt is still functional. Whereas, Cas13d requires longer crRNA, and 22-30 nt crRNA can achieve good effect. Both Cas13b and Cas13d show the ability to process precursor crRNAs. Our study suggests that Cas13b may have a stronger precursor processing ability than Cas13d. There are few in vivo studies on the application of Cas13b or Cas13d in mammals. With the methods of transgenic mice and hydrodynamic injection via tail vein, our study showed that both of them had high knock-down efficiency against target RNA in vivo. These results indicate that Cas13b and Cas13d have great potential for in vivo RNA operation and disease treatment without damaging genomic DNA.