Sars-Fang Centre, MoE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China.
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
Mol Biotechnol. 2021 Jul;63(7):613-620. doi: 10.1007/s12033-021-00324-9. Epub 2021 Apr 20.
MicroRNAs (miRNAs) regulate multicellular processes and diverse signaling pathways in organisms. The detection of the spatiotemporal expression of miRNA in vivo is crucial for uncovering the function of miRNA. However, most of the current detecting techniques cannot reflect the dynamics of miRNA sensitively in vivo. Here, we constructed a miRNA-induced CRISPR-Cas9 platform (MICR) used in marine chordate Ciona. The key component of MICR is a pre-single guide RNA (sgRNA) flanked by miRNA-binding sites that can be released by RNA-induced silencing complex (RISC) cleavage to form functional sgRNA in the presence of complementary miRNA. By using the miRNA-inducible CRISPR-on system (MICR-ON), we successfully detected the dynamic expression of a miRNA csa-miR-4018a during development of Ciona embryo. The detected patterns were validated to be consistent with the results by in situ hybridization. It is worth noting that the expression of csa-miR-4018a was examined by MICR-ON to be present in additional tissues, where no obvious signaling was detected by in situ hybridization, suggesting that the MICR-ON might be a more sensitive approach to detect miRNA signal in living animal. Thus, MICR-ON was demonstrated to be a sensitive and highly efficient approach for monitoring the dynamics of expression of miRNA in vivo and will facilitate the exploration of miRNA functions in biological systems.
微小 RNA（miRNA）在生物体内调节多细胞过程和多种信号通路。在体内检测 miRNA 的时空表达对于揭示 miRNA 的功能至关重要。然而，目前大多数检测技术无法在体内敏感地反映 miRNA 的动态变化。在这里，我们构建了一个用于海洋脊索动物海鞘的 miRNA 诱导的 CRISPR-Cas9 平台（MICR）。MICR 的关键组成部分是一个预单指导 RNA（sgRNA），其侧翼是 miRNA 结合位点，在互补 miRNA 的存在下，可被 RNA 诱导沉默复合物（RISC）切割释放，形成功能性 sgRNA。通过使用 miRNA 诱导的 CRISPR-on 系统（MICR-ON），我们成功地检测了海鞘胚胎发育过程中 miRNA csa-miR-4018a 的动态表达。检测到的模式与原位杂交的结果一致。值得注意的是，通过 MICR-ON 检测到 csa-miR-4018a 在其他组织中存在表达，而原位杂交没有检测到明显的信号，这表明 MICR-ON 可能是一种更敏感的方法来检测活体动物中的 miRNA 信号。因此，MICR-ON 被证明是一种敏感且高效的方法，可用于监测 miRNA 在体内表达的动态变化，并将有助于探索 miRNA 在生物系统中的功能。
