A Novel and Efficient Method for Bacteria Genome Editing Employing both CRISPR/Cas9 and an Antibiotic Resistance Cassette

Frontiers in Microbiology
Hong ZhangJin Wang

Abstract

As Cas9-mediated cleavage requires both protospacer and protospacer adjacent motif (PAM) sequences, it is impossible to employ the CRISPR/Cas9 system to directly edit genomic sites without available PAM sequences nearby. Here, we optimized the CRISPR/Cas9 system and developed an innovative two-step strategy for efficient genome editing of any sites, which did not rely on the availability of PAM sequences. An antibiotic resistance cassette was employed as both a positive and a negative selection marker. By integrating the optimized two-plasmid CRISPR/Cas system and donor DNA, we achieved gene insertion and point mutation with high efficiency in Escherichia coli, and importantly, obtained clean mutants with no other unwanted mutations. Moreover, genome editing of essential genes was successfully achieved using this approach with a few modifications. Therefore, our newly developed method is PAM-independent and can be used to edit any genomic loci, and we hope this method can also be used for efficient genome editing in other organisms.

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Citations

Jan 14, 2018·Applied and Environmental Microbiology·Kaifeng LiShouwen Chen
Jun 5, 2018·Biotechnology Journal·Takayuki ArazoeKeiji Nishida
Mar 25, 2019·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Gayetri Ramachandran, David Bikard
Aug 15, 2020·Applied Microbiology and Biotechnology·Chaoyong HuangYi-Xin Huo
Oct 26, 2018·Parasites & Vectors·Muhammad MajeedMona Saleh
May 6, 2020·Infection and Drug Resistance·Pourya GholizadehHossein Samadi Kafil
Oct 26, 2018·Frontiers in Microbiology·Xiang AoYingying Guo
May 26, 2020·BMC Microbiology·Francesca Romana CianfanelliDirk Bumann
Jun 26, 2020·World Journal of Microbiology & Biotechnology·Atieh Hashemi
Mar 7, 2021·Microorganisms·Mouna HamelSophie Alexandra Baron

Methods Mentioned

BETA
genetic modification
PCR
fluorescence microscopy
FACS
electrophoresis
flow cytometry

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