title: Small indels induced by CRISPR/Cas9 in the 5' region of microRNA lead to its depletion and Drosha processing retardance.
abstract: MicroRNA knockout by genome editing technologies is promising. In order to extend the application of the technology and to investigate the function of a specific miRNA, we used CRISPR/Cas9 to deplete human miR-93 from a cluster by targeting its 5' region in HeLa cells. Various small indels were induced in the targeted region containing the Drosha processing site and seed sequences. Interestingly, we found that even a single nucleotide deletion led to complete knockout of the target miRNA with high specificity. Functional knockout was confirmed by phenotype analysis. Furthermore, de novo microRNAs were not found by RNA-seq. Nevertheless, expression of the pri-microRNAs was increased. When combined with structural analysis, the data indicated that biogenesis was impaired. Altogether, we showed that small indels in the 5' region of a microRNA result in sequence depletion as well as Drosha processing retard.
authors.affiliation: a Institute of Molecular Medicine ; Peking University ; Beijing , PR China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Jiang
authors.forename: Xing
authors.initials: X
authors.lastname: Meng
authors.forename: Lingwei
authors.initials: L
authors.lastname: Meng
authors.forename: Nannan
authors.initials: N
authors.lastname: Chang
authors.forename: Jingwei
authors.initials: J
authors.lastname: Xiong
authors.forename: Huiqing
authors.initials: H
authors.lastname: Cao
authors.forename: Zicai
authors.initials: Z
authors.lastname: Liang
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D049109:Cell Proliferation; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D054643:INDEL Mutation / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism
chemical_list: C543350:MIRN93 microRNA, human; D035683:MicroRNAs; D012322:RNA Precursors; D012333:RNA, Messenger; C517918:DROSHA protein, human; D043244:Ribonuclease III
keywords: CRISPR, clustered, regularly interspaced, short palindromic repeats; CRISPR/Cas9; PAM, the protospacer adjacent motif; RNA-guided DNA cleavage; crRNA, CRISPR RNAs; miR-93; microRNA biogenesis; microRNA depletion