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Effective CRISPRa-Mediated Control of Gene Expression in Bacteria Must Overcome Strict Target Site Requirements
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Effective CRISPRa-Mediated Control of Gene Expression in Bacteria Must Overcome Strict Target Site Requirements
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Paper
Effective CRISPRa-Mediated Control of Gene Expression in Bacteria Must Overcome Strict Target Site Requirements
2019
Overview
Abstract In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we currently lack a complete understanding of the rules for designing effective guide RNA target sites. We have identified multiple features of bacterial promoters that impose stringent requirements on bacterial CRISPRa target sites. Most importantly, we found that shifting a gRNA target site by 2-4 bases along the DNA target can cause a nearly complete loss in activity. The loss in activity can be rescued by shifting the target site 10-11 bases, corresponding to one full helical turn. Practically, our results suggest that it will be challenging to find a gRNA target site with an appropriate PAM sequence at precisely the right position at arbitrary genes of interest. To overcome this limitation, we demonstrate that a dCas9 variant with expanded PAM specificity allows activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.
Publisher
Cold Spring Harbor Laboratory Press,Cold Spring Harbor Laboratory
Subject
