[Source: Journal of Virology, full page: (LINK). Abstract, edited.]
Efficient genome engineering of a virulent Klebsiella bacteriophage using CRISPR-Cas9
Juntao Shen1, Jinjie Zhou1, Guo-Qiang Chen2 and Zhi-Long Xiu1⇑
Author Affiliations: 1 School of Life Science and Biotechnology, Dalian University of Technology, Dalian, PR China; 2 School of Life Sciences, Tsinghua University, Beijing, PR China
ABSTRACT
Klebsiella pneumoniae is one of the most common nosocomial opportunistic pathogens usually with multiple drug-resistance. Phage therapy, a potential new therapeutics to replace or supplement antibiotics, has attracted much attention. However, very few Klebsiella phages have been well-characterized as the lack of efficient genome editing tools. Here, Cas9 from Streptococcus pyogenes and a single guide RNA (sgRNA) were used to modify a virulent Klebsiella bacteriophage phiKpS2. We firstly evaluated the distribution of sgRNA activity in phages and proved that it’s largely inconsistent with the predicted activity from current models trained on eukaryotic cell datasets. A simple CRISPR-based phage genome editing procedure was developed based on the discovery that homologous arms as short as 30-60 bp was sufficient to introduce point mutation, gene deletion and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the above strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy.
IMPORTANCE
In the present study, we have addressed an efficient, time-saving and cost-effective CRISPR-based phage genome editing of Klebsiella phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote the applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proved enough to introduce point mutation, small frameshift deletion, gene deletion and swap into phages, and weak sgRNAs were proved useful for precise phage genome editing but failed to select random recombinants, which all make the CRISPR-based phage genome editing easier to use.
FOOTNOTES
Corresponding author: Prof. Dr. Zhi-Long Xiu, School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, PR China., E-mail: zhlxiu@dlut.edu.cn, Tel: +86-0411-84706369
Copyright © 2018 American Society for Microbiology. All Rights Reserved.
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Keywords: Antibiotics; Drugs Resistance; Klebsiella pneumoniae; Bacteriophages; CRISPR-Cas9.
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