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. 2021 Nov 23;9(12):2414. doi: 10.3390/microorganisms9122414

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Construction of a DNA Encoded and Displayed Antigens of SARS-CoV-2 (DEaDAS) recombinant phage. (A) The integrase gene (blue) of phage phiTM45 was replaced with a mammalian expression cassette (gray box) containing the RBD of SARS-CoV2 (aqua) driven by a CMV promoter (pCMV). The location of the sgRNA target selecting against parent phiTM45 (green) is indicated. Regions of homology between the synthetic substrate and phiTM45 are indicated (blue line), and the structure of recombinant phage DEaDAS-1 is shown below. (B) sgRNA induction for selection against the integrase gene reduces Bxb1 and phiTM45 plaquing by at least two orders of magnitude. (C) Primary plaques (left panel) recovered from CRISPY-BRED engineering were screened by PCR, and a candidate recombinant (candidate 2) was plaque purified and secondary plaques (right panel) re-screened by PCR; all candidates are recombinant.