Establishment of novel mutagenesis revealing evolutionarily functional miRNAs locus

Genomic research has achieved to reveal how genomes regulate biological phenomena. In order to understand functions of genomes, it is a powerful approach to evaluate loss of function of them under biological phenomena. In fact, functional analyses have been conducted on coding sequences rather than non-coding sequences. The main reason is that it is not straightforward to determine functionally critical regions affecting phenotypes due to no reading frame. Whole genomic random mutagenesis and deletion of whole candidate regions could induce loss of function of non-coding sequences but could not identify definite functionally critical regions. Therefore, to identify functionally critical regions and conduct functional analyses on non-coding sequences in a biological context, it requires to develop a genomic approach into site-specific rather than genome-wide mutagenesis. Here, we established novel site-specific random mutagenesis named CRISPR & Transposase based RegionaL Mutagenesis (CTRL-Mutagenesis). We clearly demonstrated that CTRL-mutagenesis randomly induce diverse mutations within only target regions in murine embryonic stem cells (mES cells). This random mutant mES clone library could expand to functional analyses under in vitro and in vivo biological development. Comparative analysis in this mutant library harbouring subtly different mutations within only the same region would be effective for tackling these challenges.