Title: Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems

Abstract

The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report heremore » the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.« less

Authors:

Liu, Hualan  ^[1];

• Search DOE PAGES for author "Liu, Hualan"
• Search DOE PAGES for ORCID "0000-0002-9636-3855"

Robinson, David S. ^[2]; Wu, Zong-Yen ^[3]; Kuo, Rita ^[2]; Yoshikuni, Yasuo ^[1]; Blaby, Ian K. ^[1]; Cheng, Jan-Fang ^[1]

---

+ Show Author Affiliations

1. USDOE Joint Genome Institute (JGI), Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
2. USDOE Joint Genome Institute (JGI), Berkeley, CA (United States)
3. USDOE Joint Genome Institute (JGI), Berkeley, CA (United States); National Chung Hsing Univ., Taichung (Taiwan)

Publication Date:

Wed Nov 04 00:00:00 EST 2020

Research Org.:

Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC)

OSTI Identifier:

1764518

Grant/Contract Number:  

AC02-05CH11231

Resource Type:

Accepted Manuscript

Journal Name:

PLoS ONE

Additional Journal Information:

Journal Volume: 15; Journal Issue: 11; Journal ID: ISSN 1932-6203

Publisher:

Public Library of Science

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Polymerase chain reactions; CRISPR; genomics; DNA repair; bacterial genomics; chromosomal deletions; transposable elements; plasmid construction