Genetic manipulation of Bacteriophage T4 utilizing the CRISPR-Cas13b system

Abstract

ABSTRACTCRISPR-Cas type II and type V systems are inefficient in modifying bacteriophage T4 genome, due to the hypermodification of its DNA. Here, we present a genome editing technique for bacteriophage T4 using the type VI CRISPR-Cas system. Using BzCas13b targeting of T4 phage, we were able to individually delete both T4 glucosyl transferase genes,α-gtandβ-gt. Furthermore, we employed this method to mutate two conserved residues within the T4 DNA polymerase and to introduce the yellow fluorescent protein (YFP) coding sequence into T4 phage genome, enabling us to visualize phage infections. The T4 genome editing protocol was optimized to obtain recombinant phages within a 6-hour timescale. Finally, spacers homologous to a variety of T4 genes were used to study the generality of Cas13b targeting, revealing important variability in targeting efficiency. Overall, this study presents a detailed description of the rapid and easy production of T4 phage specific mutants.IMPORTANCEThe use of phages for therapeutic purposes requires a complete understanding of their life cycle. For this purpose, it’s very useful to have a wide range of phage genome engineering tools at our disposal, each adapted to a particular phage or situation. Although T4 phage has been studied extensively over the past seven decades, a complete understanding of its lytic cycle is still lacking. Cas9- and Cpf1-dependent genome editing techniques for T4 have proven to be inconsistent due to the glucosyl-hydroxymethyl modification of the cytosine residues in its genome. The RNA targeting of the Cas13 system presents an ideal alternative, as demonstrated here, to overcome interference from DNA hypermodification. Apart from demonstrating a new genome editing technique in T4, we have generated a range of T4 variants demonstrating the efficacy of our technique in obtaining meaningful and difficult to construct phage mutants.