A bacterial gene-drive system efficiently edits and inactivates a high copy number antibiotic resistance locus

J Andrés Valderrama,Surashree S Kulkarni,Ethan Bier,Victor Nizet

doi:10.1038/s41467-019-13649-6

J Andrés Valderrama, Surashree S Kulkarni + Show 2 more

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https://doi.org/10.1038/s41467-019-13649-6

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Journal: Nature Communications	Publication Date: Dec 1, 2019
Citations: 45	License type: open-access

Affiliation: University of California, San Diego

Abstract

Gene-drive systems in diploid organisms bias the inheritance of one allele over another. CRISPR-based gene-drive expresses a guide RNA (gRNA) into the genome at the site where the gRNA directs Cas9-mediated cleavage. In the presence of Cas9, the gRNA cassette and any linked cargo sequences are copied via homology-directed repair (HDR) onto the homologous chromosome. Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas9 cleavage site. This “pro-active” genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. Pro-AG can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine.

Highlights

Gene-drive systems in diploid organisms bias the inheritance of one allele over another
As copying of the gRNA2 expression cassette from a low to a high copy number plasmid significantly amplified guide RNA (gRNA) gene number and expression levels, we wondered whether the enhanced performance of pro-active genetics (Pro-AG) versus CRISPR-control could be wholly attributable to this effect. We addressed this question by comparing ampicillin resistance (AmpR) colony-forming unit (CFU) reduction using CRISPR-control versus Pro-AG configurations in a situation where the bla-targeting gRNA2 was encoded from the outset on a high copy number plasmid, namely the pETg target plasmid itself, which would limit gRNA amplification to at most twofold
Cumulatively, our results indicate that the Pro-AG configuration is 2–3 orders of magnitude more efficient in disrupting the activity of a high copy number target gene than the CRISPRcontrol arrangement, yielding to a 4–5 log10-fold reduction in AmpR E. coli, fully attributed to precise insertion of the gRNA (± cargo) cassette from the editing vector into the targeted gRNA cleavage site

Summary

Introduction

Gene-drive systems in diploid organisms bias the inheritance of one allele over another. We develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas[9] cleavage site This “proactive” genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. When doublestranded DNA breaks are induced in the germline by the gRNA/ Cas[9] complex, the gRNA together with any linked cargo sequences are copied into the break via the homologydirected repair (HDR) pathway[6] Such gene conversion events can greatly bias transmission of the gRNA cassette so that it is inherited by most progeny. Pro-AG expands the available toolkit for engineering or manipulating bacteria in future biotechnology and biomedicine applications

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