Zheng Linggang, Tan Yang, Hu Yucan, Shen Juntao, Qu Zepeng, Chen Xianbo, Ho Chun Loong, Leung Elaine Lai-Han, Zhao Wei, Dai Lei
CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Shenzhen 518055, China.
Dr. Neher's Biophysics Laboratory for Innovative Drug Discovery/State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macau 999078, China.
ACS Synth Biol. 2022 Jan 21;11(1):464-472. doi: 10.1021/acssynbio.1c00543. Epub 2022 Jan 6.
is the most abundant genus in the human gut microbiome and has been increasingly used as model organisms for studying the function and ecology of the gut microbiome. However, genome editing tools for such commensal gut microbes are still lacking. Here we developed a versatile, highly efficient CRISPR/Cas-based genome editing tool that allows markerless gene deletion and insertion in human gut species. We constructed multiple CRISPR/Cas systems in all-in-one - shuttle plasmids and systematically evaluated the genome editing efficiency in , including the mode of Cas protein expression (constitutive, inducible), different Cas proteins (FnCas12a, SpRY, SpCas9), and sgRNAs. Using the anhydrotetracycline (aTc)-inducible CRISPR/FnCas12a system, we successfully deleted large genomic fragments up to 50 kb to study the function of metabolic gene clusters. Furthermore, we demonstrated that CRISPR/FnCas12a can be broadly applied to engineer multiple human gut species, including , , , and . We envision that CRISPR/Cas-based genome editing tools for will greatly facilitate mechanistic studies of the gut commensal and the development of engineered live biotherapeutics.
ACS Synth Biol. 2022-1-21
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