GSE130510
Disruption of miRNA Sequence by TALENs and CRISPR/Cas9 Induces Varied Length of miRNA Production [CRISPR/Cas9]
Purpose: MicroRNAs (miRNAs) have been recognized as crucial players in plant growth, development, production and responses to biotic and abiotic stresses, while only limited number of mutants can be used to dissect their roles. Here we used TALENs (Transcription Activator-Like Effector Nucleases) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to introduce base pair mutations in MIRNA genes, resulting in creation of miRNA mutants and elucidating the effect of mutations on the miRNA biogenesis in rice and Arabidopsis. Methods: Total RNAs were prepared using a Trizol reagent-based method. The plant materials were homogenized and reconstituted in Trizol solution. The solution in turn was extracted by chloroform. Isopropanol was used to precipitate the total RNA. The whole process was performed under RNase free environment. DEPC-treated water was used to dissolve RNA. The total RNA underwent miRNA isolation using Qiagen miRNA purification kit according to the manufacturer's manual. Small RNA libraries were made using 5 µg total RNA with Illumina TruSeq Small RNA Library Prep Kit as described, and sequenced on the Illumina HiSeq 2500 platform. Overall design: To study the change of Arabidopsis siRNAs by CRISPR/Cas9-mediated mutations.