Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify
Kento Morimoto1,2, Hayate Suzuki3, Akihiro Kuno4
1Doctoral Program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
Open Biology
|April 2, 2024
Summary
A new CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis) method enables targeted mutations in specific genomic regions. This advances the study of cis-elements and microRNA clusters.
Area of Science:
- Genomics
- Molecular Biology
- Gene Regulation
Background:
- Functional genomic regions are crucial for biological processes but challenging to study.
- Existing mutagenesis methods are insufficient for analyzing cis-elements and microRNA clusters.
- There is a need for tools to specifically target and mutate these non-coding regions.
Purpose of the Study:
- To establish a novel region-specific random mutagenesis method.
- To enable functional analysis of cis-elements and microRNA clusters.
- To facilitate the study of complex gene regulatory elements.
Main Methods:
- Development of CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis).
- Application of CTRL-mutagenesis in murine embryonic stem cells.
- Induction of random, diverse mutations within targeted genomic regions.
Main Results:
- CTRL-mutagenesis successfully induced diverse mutations in target regions.
- The method demonstrated region-specific mutagenesis.
- Generated mutants are suitable for comparative analysis of cis-elements and microRNA clusters.
Conclusions:
- CTRL-mutagenesis is a novel and effective tool for regional mutagenesis.
- This method overcomes limitations of previous mutagenesis approaches.
- Facilitates in-depth functional studies of cis-elements and microRNA clusters.
