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

Abstract

Summary: 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). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Biotechnology and bioengineering
• CRISPR
• Genetics
• Microbiology