RT Journal Article
SR Electronic
T1 A novel prokaryotic CRISPR-Cas12a based tool for programmable transcriptional activation and repression
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.08.05.232744
DO 10.1101/2020.08.05.232744
A1 Christoph Schilling
A1 Mattheos Koffas
A1 Volker Sieber
A1 Jochen Schmid
YR 2020
UL http://biorxiv.org/content/early/2020/08/05/2020.08.05.232744.abstract
AB Transcriptional perturbation using inactivated CRISPR-nucleases (dCas) is a common method in eukaryotic organisms. While rare examples of dCas9 based tools for prokaryotes have been described, multiplexing approaches are limited due to the used effector nuclease. For the first time, a dCas12a derived broad host range tool for the targeted activation and repression of genes was developed. Therefore, a previously described SoxS activator domain was linked to dCas12a to enable programmable activation of gene expression. Proof of principle of transcriptional regulation was demonstrated based on fluorescence reporter assays using the alternative host organism Paenibacillus polymyxa as well as Escherichia coli. Single target and multiplex CRISPR interference targeting the exopolysaccharide biosynthesis of P. polymyxa was shown to emulate polymer compositions of gene knock-outs. Simultaneous expression of 11 gRNAs targeting multiple lactate dehydrogenases and a butanediol dehydrogenase resulted in decreased lactate formation, as well as an increased butanediol production in microaerobic fermentation processes. Even though Cas12a is more restricted in terms of its genomic target sequences compared to Cas9, its ability to efficiently process its own guide RNAs in vivo makes it a promising tool to orchestrate sophisticated genetic reprogramming of bacterial cells or to screen for engineering targets in the genome. The developed tool will accelerate metabolic engineering efforts in common synthetic bacterial cell factories such as E. coli, as well as promising alternative host organisms.Competing Interest StatementThe authors have declared no competing interest.CRISPRclustered regularly interspaced short palindromic repeatsCRISPRiCRISPR interferenceCRISPRaCRISPR activationgRNAguide RNAGTiinitiating glycosyltransferase2,3-BDL2,3-butanediolEPSexopolysaccharide