Zhu Ruiying, Ren Chuanhong, Bao Zehua
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310058, Zhejiang, China.
Zhejiang Key Laboratory of Intelligent Manufacturing for Functional Chemicals, ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, 311215, Zhejiang, China.
Genome Biol. 2025 Sep 23;26(1):297. doi: 10.1186/s13059-025-03756-7.
Gene diversification is an effective approach to massively dissecting variant functions and evolving sequences when paired with an appropriate assay. In vitro mutagenesis and ectopic gene expression, however, fail to simulate the endogenous regulatory environment of the variants. The development of clustered, regularly interspaced short palindromic repeats (CRISPR) systems has greatly boosted the efficiency of targeted gene diversification in various species. Here, we review recent CRISPR-assisted methods for chromosomal gene diversification and artificial evolution, focusing on the advantages and limitations of each approach, and propose possible strategies to overcome current limitations and directions in future technology development.
