Chromosomal integration of complex DNA constructs using CRAGE and CRAGE-Duet systems

Zhiying Zhao; Jan-Fang Cheng; Yasuo Yoshikuni

doi:10.1016/j.xpro.2022.101546

Chromosomal integration of complex DNA constructs using CRAGE and CRAGE-Duet systems

STAR Protoc. 2022 Sep 16;3(3):101546. doi: 10.1016/j.xpro.2022.101546. Epub 2022 Jul 16.

Authors

Zhiying Zhao¹, Jan-Fang Cheng², Yasuo Yoshikuni³

Affiliations

¹ The US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: zyzhao@lbl.gov.
² The US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
³ The US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Center for Advanced Bioenergy and Bioproducts Innovation, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Global Center for Food, Land, and Water Resources, Hokkaido University, Hokkaido 060-8589, Japan. Electronic address: yyoshikuni@lbl.gov.

Abstract

Our recent development of the CRAGE (chassis-independent recombinase-assisted genome engineering) system enables single-step integration of large, complex DNA constructs directly into bacteria genomes across multiple phyla. This protocol describes the details of the experimental design and procedures of CRAGE and extended CRAGE-Duet systems. It also describes a strategy that combines CRISPR with CRAGE, which allows implementation of CRISPR-Cas9, CRISPRa, and CRISPRi in diverse bacteria, overcoming major limitations to broaden the application of CRISPR in non-model bacterial genome engineering. For complete details on the use and execution of this protocol, please refer to Wang et al. (2019), Wang et al. (2020), and Liu et al. (2020).

Keywords: Biotechnology and bioengineering; CRISPR; Genetics; Microbiology.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Bacteria
DNA
Genome, Bacterial*
Recombinases*

Substances

Recombinases
DNA