Abstract
Traditional low-efficiency homologous recombination between plasmids and phage genome was developed to generate recombinant phages with gene replacement, deletion, or insertion. Here, we describe a protocol for well-known Streptococcus pyogenes CRISPR-Cas9-mediated genome editing of lytic Escherichia coli T4 phage. We introduce two methods for point mutation that yield a BamHI restriction site in the target region and a fluorescent protein tag (GFP) insertion in T4 phage genome as examples. Engineered T4 phages were obtained in 100% efficiency. This protocol can be adapted for any other phage modifications by active heterologous CRISPR-Cas9 in their host.
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© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Chen, Y., Li, X., Wang, S., Qian, P., Li, Y. (2021). CRISPR-Cas9-Mediated Genome Editing in Escherichia coli Bacteriophages. In: Islam, M.T., Molla, K.A. (eds) CRISPR-Cas Methods. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1657-4_21
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DOI: https://doi.org/10.1007/978-1-0716-1657-4_21
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1656-7
Online ISBN: 978-1-0716-1657-4
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