CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli

Abstract

Governance of the endogenous gene regulatory network enables the navigation of cells towards beneficial traits for recombinant protein production. CRISPRactivation and interference provides the basis for gene expression modulation but is primarily applied in eukaryotes. Particularly the lack of wide-ranging prokaryotic CRISPRa studies might be attributed to intrinsic limitations of bacterial activators and Cas9 proteins. While bacterial activators need accurate spatial orientation and distancing towards the target promoter to be functional, Cas9-based CRISPR tools only bind sites adjacent to NGG PAM sequences. These circumstances hampered Cas9-guided activators from mediating the up-regulation of endogenous genes at precise positions in bacteria. We could overcome this limitation by combining the PAM independent Cas9 variant SpRY and a CRISPRa construct using phage protein MCP fused to transcriptional activator SoxS. This CRISPRa construct, referred to as SMS, was compared with previously reported CRISPRa constructs and showed up-regulation of a reporter gene library independent of its PAM sequence in Escherichia coli. We also demonstrated down-regulation and multi-gene expression control with SMS at non-NGG PAM sites. Furthermore, we successfully applied SMS to up-regulate endogenous genes, and transgenes at non-NGG PAM sites, which was impossible with the previous CRISPRa construct.

Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible dCas9 Variants

“…While this manuscript was under revision, another manuscript was published that reported broadly consistent findings with the dSpRY variant. 35 Our work includes additional comparisons with multiple PAM-flexible dCas9 variants and identifies situations where some of these variants (notably dxCas9-NG) are more effective than dSpRY. We also observed trade-offs with CRISPRi efficiency that have not been previously described.…”

From DNA-protein interactions to the genetic circuit design using CRISPR-dCas systems

“…A number of such activator domains have been used so far, including the omega subunit of RNAP, TetD, and SoxS activators from E. coli ( Dong et al, 2018 ; Villegas Kcam et al, 2021 ). SoxS-based designs have been found to confer the best results, yielding up to a 50-fold increase in mRNA levels of activated genes, provided the SoxS domain has proper positioning and orientation relative to the promoter ( Dong et al, 2018 ; Klanschnig et al, 2022 ).…”

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

“…Therefore, powerful and efficient genetic manipulation methods for Xanthomonas are always in great demand. In the past decade, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)mediated genome editing technology, derived from many bacteria and most archaea, has revolutionized everything from basic science to translational applications related to microorganisms, plants, animals, and human beings [3][4][5][6][7][8]. However, the application of CRISPR technology in Xanthomonas species, or even in plant pathogenic bacteria, has rarely been studied.…”