Authors
Guanhua Yuan, Zhe Gao, Yingxuan Qi, Yang Zhang, Xuhui Tian, Pengpeng Zhao, Xu Feng, Qunxin She
Published in
Advanced science (Weinheim, Baden-Wurttemberg, Germany). Pages e76042. Jun 29, 2026. Epub Jun 29, 2026.
Abstract
The construction of minimal-genome microbes offers an ideal platform for understanding fundamental biological processes and synthetic biology, yet the research is hindered by incomplete lists of essential genes in microbes and by multiple rounds of genome trimming with a trial-and-error nature. To address this, we introduce CREAT (CRISPR-based genome trimming with a multi-homology-arm template)-a streamlined approach that integrates CRISPR-targeted genome cleavage and homology arm walking to classify essential from non-essential genomic subregions, thus providing the basis for predicting essential genes in a given organism. These essential genes were then assembled into synthetic gene cassettes for one-step replacement of the targeted non-deletable genomic regions for further genome trimming. Eight consecutive rounds of CREAT genome trimming achieved a 20.8% reduction in genome size in Saccharolobus islandicus. Furthermore, Cas9-based CREAT genome trimming was developed for Bacillus subtilis and Escherichia coli, with efficiency greatly enhanced by the λ-Red recombinase in the latter. Together, this iterative application of CREAT provides a scalable and generally applicable strategy for rapidly constructing minimal genomes across diverse microorganisms.
PMID:
42371672
Bibliographic data and abstract were imported from PubMed on 29 Jun 2026.
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