title: Gene transcription repression in Clostridium beijerinckii using CRISPR-dCas9.
abstract: CRISPR-Cas9 has been explored as a powerful tool for genome engineering for many organisms. Meanwhile, dCas9 which lacks endonuclease activity but can still bind to target loci has been engineered for efficient gene transcription repression. Clostridium beijerinckii, an industrially significant species capable of biosolvent production, is generally difficult to metabolically engineer. Recently, we reported our work in developing customized CRISPR-Cas9 system for genome engineering in C. beijerinckii. However, in many cases, gene expression repression (rather than actual DNA mutation) is more desirable for various biotechnological applications. Here, we further demonstrated gene transcription repression in C. beijerinckii using CRISPR-dCas9. A small RNA promoter was employed to drive the expression of the single chimeric guide RNA targeting on the promoter region of amylase gene, while a constitutive thiolase promoter was used to drive Streptococcus pyogenes dCas9 expression. The growth assay on starch agar plates showed qualitatively significant repression of amylase activity in C. beijerinckii transformant with CRISPR-dCas9 compared to the control strain. Further amylase activity quantification demonstrated consistent repression (65-97% through the fermentation process) on the activity in the transformant with CRISPR-dCas9 versus in the control. Our results provided essential references for engineering CRISPR-dCas9 as an effective tool for tunable gene transcription repression in diverse microorganisms. Biotechnol. Bioeng. 2016;113: 2739-2743. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois.
authors.forename: Zhong-Tian
authors.initials: ZT
authors.lastname: Zhang
authors.affiliation: Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois.
authors.forename: Seung-Oh
authors.initials: SO
authors.lastname: Seo
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois.
authors.forename: Patrick
authors.initials: P
authors.lastname: Lynn
authors.affiliation: Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois.|Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois.
authors.forename: Ting
authors.initials: T
authors.lastname: Lu
authors.affiliation: Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois.
authors.forename: Yong-Su
authors.initials: YS
authors.lastname: Jin
authors.affiliation: Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois. blaschek@illinois.edu.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois. blaschek@illinois.edu.|The Integrated Bioprocessing Research Laboratory (IBRL), University of Illinois at Urbana-Champaign, 1207 W Gregory Drive, Urbana, 61801, Illinois. blaschek@illinois.edu.
authors.forename: Hans P
authors.initials: HP
authors.lastname: Blaschek
mesh_terms: D000681:Amylases / Q000096:biosynthesis / Q000235:genetics*; D064130:CRISPR-Associated Proteins / Q000235:genetics; D046970:Clostridium beijerinckii / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics; D063185:Epigenetic Repression / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D000681:Amylases
keywords: CRISPR-dCas9; CRISPRi; amylase activity; gene transcription repression; genome engineering; synthetic biology