No abstract

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 tool for the targeted activation and repression of genes was developed. Therefore, a previously described SoxS activator domain was linked to dCas12a to enable the programmable activation of gene expression. A proof of principle of transcriptional regulation was demonstrated on the basis of 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 knockouts. The 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 the alternative host organism P. polymyxa and might be also applied for other bacterial cell factories.

Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome engineering and related technologies have revolutionized biotechnology over the last decade by enhancing the efficiency of sophisticated biological systems. Cas12a (Cpf1) is an RNA-guided endonuclease associated to the CRISPR adaptive immune system found in many prokaryotes. Contrary to its more prominent counterpart Cas9, Cas12a recognizes A/T rich DNA sequences and is able to process its corresponding guide RNA directly, rendering it a versatile tool for multiplex genome editing efforts and other applications in biotechnology. While Cas12a has been extensively used in eukaryotic cell systems, microbial applications are still limited. In this review, we highlight the mechanistic and functional differences between Cas12a and Cas9 and focus on recent advances of applications using Cas12a in bacterial hosts. Furthermore, we discuss advantages as well as current challenges and give a future outlook for this promising alternative CRISPR-Cas system for bacterial genome editing and beyond. Key points • Cas12a is a powerful tool for genome engineering and transcriptional perturbation • Cas12a causes less toxic side effects in bacteria than Cas9 • Self-processing of crRNA arrays facilitates multiplexing approaches