Title: CRISPR-based engineering of phages for in situ bacterial base editing

Abstract

Investigation of microbial gene function is essential to the elucidation of ecological roles and complex genetic interactions that take place in microbial communities. While microbiome studies have increased in prevalence, the lack of viable in situ editing strategies impedes experimental progress, rendering genetic knowledge and manipulation of microbial communities largely inaccessible. Here, we demonstrate the utility of phage-delivered CRISPR-Cas payloads to perform targeted genetic manipulation within a community context, deploying a fabricated ecosystem (EcoFAB) as an analog for the soil microbiome. First, we detail the engineering of two classical phages for community editing using recombination to replace nonessential genes through Cas9-based selection. We show efficient engineering of T7, then demonstrate the expression of antibiotic resistance and fluorescent genes from an engineered λ prophage within an Escherichia coli host. Next, we modify λ to express an APOBEC-1-based cytosine base editor (CBE), which we leverage to perform C-to-T point mutations guided by a modified Cas9 containing only a single active nucleolytic domain (nCas9). We strategically introduce these base substitutions to create premature stop codons in-frame, inactivating both chromosomal ( lacZ ) and plasmid-encoded genes (mCherry and ampicillin resistance) without perturbation of the surrounding genomic regions. Furthermore, using a multigenera synthetic soil community,more » we employ phage-assisted base editing to induce host-specific phenotypic alterations in a community context both in vitro and within the EcoFAB, observing editing efficiencies from 10 to 28% across the bacterial population. The concurrent use of a synthetic microbial community, soil matrix, and EcoFAB device provides a controlled and reproducible model to more closely approximate in situ editing of the soil microbiome.« less

Authors:

Nethery, Matthew A. ^[1]; Hidalgo-Cantabrana, Claudio  ^[2];

• Search DOE PAGES for author "Hidalgo-Cantabrana, Claudio"
• Search DOE PAGES for ORCID "0000-0002-7248-4564"

Roberts, Avery ^[1]; Barrangou, Rodolphe  ^[1]

• Search DOE PAGES for author "Barrangou, Rodolphe"
• Search DOE PAGES for ORCID "0000-0002-0648-3504"

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+ Show Author Affiliations

1. Genomic Sciences Graduate Program, North Carolina State University, Raleigh, NC 27695, Department of Food, Bioprocessing &, Nutrition Sciences, North Carolina State University, Raleigh, NC 27606
2. Department of Food, Bioprocessing &, Nutrition Sciences, North Carolina State University, Raleigh, NC 27606

Publication Date:

Mon Nov 07 00:00:00 EST 2022

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1897298

Alternate Identifier(s):

OSTI ID: 1897669

Grant/Contract Number:  

AC02-05CH11231

Resource Type:

Published Article

Journal Name:

Proceedings of the National Academy of Sciences of the United States of America

Additional Journal Information:

Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 119 Journal Issue: 46; Journal ID: ISSN 0027-8424

Publisher:

Proceedings of the National Academy of Sciences

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; phage engineering; CRISPIR; base editing; soil microbiome