Genome Engineering of Virulent Lactococcal Phages Using CRISPR-Cas9

Abstract

Phages are biological entities found in every ecosystem. Although much has been learned about them in past decades, significant knowledge gaps remain. Manipulating virulent phage genomes is challenging. To date, no efficient gene-editing tools exist for engineering virulent lactococcal phages. Lactococcus lactis is a bacterium extensively used as a starter culture in various milk fermentation processes, and its phage sensitivity poses a constant risk to the cheese industry. The lactococcal phage p2 is one of the best-studied models for these virulent phages. Despite its importance, almost half of its genes have no functional assignment. CRISPR-Cas9 genome editing technology, which is derived from a natural prokaryotic defense mechanism, offers new strategies for phage research. Here, the well-known Streptococcus pyogenes CRISPR-Cas9 was used in a heterologous host to modify the genome of a strictly lytic phage. Implementation of our adapted CRISPR-Cas9 tool in the prototype phage-sensitive host L. lactis MG1363 allowed us to modify the genome of phage p2. A simple, reproducible technique to generate precise mutations that allow the study of lytic phage genes and their encoded proteins in vivo is described.

CRISPR–Cas9 Based Bacteriophage Genome Editing

“…When the exogenous Streptococcus pyogenes SF370 II-A system (CRISPR–Cas9 system) is used to perform gene editing of phage TT4P2 in V. natriegens TT4, it is necessary to determine whether the edited phage harbors anti-Cas9 proteins or an endogenous CRISPR–Cas system, which may cause SpyCas9:RNA inactivity, meaning that it cannot be used as a screening tool ( 53 – 55 ). Compared with three other cases of editing phage genes using the CRISPR–Cas9 system, the effect of SpyCas9:RNA on the random mutation selection of phage target sequences and their PAM in this study was similar to that in the study of Lemay ( 43 ), which found almost no point mutations in the target sequence and its PAM among single plaques. However, the subsequent orf6 deletion experiments in this study showed that the editing efficiency was higher than that in the Lemay study and was similar to those in the studies by Tao ( 44 ) and Shen ( 45 ), at approximately 100%.…”

“…However, according to research by Lemay ( 43 ) and Shen ( 45 ) and the results of this study, if the activity of exogenous SpyCas9:RNA is not high and the strain containing the targeted plasmid has a lower effect on the selection of random mutations, then the editing efficiency of the double-plasmid strain for the targeted phage gene depends on the specific situation. For example, the editing efficiency of the nonessential gene orf6 in this study was nearly 100%, and the mutants were all homozygous.…”

CRISPR–Cas9 Based Bacteriophage Genome Editing

“…When the exogenous Streptococcus pyogenes SF370 II-A system (CRISPR–Cas9 system) is used to perform gene editing of phage TT4P2 in V. natriegens TT4, it is necessary to determine whether the edited phage harbors anti-Cas9 proteins or an endogenous CRISPR–Cas system, which may cause SpyCas9:RNA inactivity, meaning that it cannot be used as a screening tool ( 53 – 55 ). Compared with three other cases of editing phage genes using the CRISPR–Cas9 system, the effect of SpyCas9:RNA on the random mutation selection of phage target sequences and their PAM in this study was similar to that in the study of Lemay ( 43 ), which found almost no point mutations in the target sequence and its PAM among single plaques. However, the subsequent orf6 deletion experiments in this study showed that the editing efficiency was higher than that in the Lemay study and was similar to those in the studies by Tao ( 44 ) and Shen ( 45 ), at approximately 100%.…”

“…However, according to research by Lemay ( 43 ) and Shen ( 45 ) and the results of this study, if the activity of exogenous SpyCas9:RNA is not high and the strain containing the targeted plasmid has a lower effect on the selection of random mutations, then the editing efficiency of the double-plasmid strain for the targeted phage gene depends on the specific situation. For example, the editing efficiency of the nonessential gene orf6 in this study was nearly 100%, and the mutants were all homozygous.…”

Rapid detection of <italic>Escherichia coli</italic> in bloodstream infection via CRISPR-Cas9 engineered reporter phage T7::<italic>Nluc</italic> and microfluidic chip platform

“…In contrast to conventional plaque screening, this method allows the rapid identification of recombinants. Interestingly, we observed heterozygous PCR bands representing both T7 and T7:: Nluc within individual plaques, a phenomenon previously reported in low-activity sgRNA contexts [ 31 , 36 ], but rarely observed with high-affinity sgRNAs. The ELISA approach circumvented this complication, enhancing the overall screening efficiency.…”

Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

“…4d). The phenomenon has also been observed in L. lactis for genome editing of lactococcal phage p2 (16). A possible explanation of those results is that inefficient cleavage can be tolerated through FIG 5 Construction of homologous recombination templates for point mutation, gene deletion, and swap.…”

CRISPR–Cas9 Based Bacteriophage Genome Editing

“…When the exogenous Streptococcus pyogenes SF370 II-A system (CRISPR–Cas9 system) is used to perform gene editing of phage TT4P2 in V. natriegens TT4, it is necessary to determine whether the edited phage harbors anti-Cas9 proteins or an endogenous CRISPR–Cas system, which may cause SpyCas9:RNA inactivity, meaning that it cannot be used as a screening tool ( 53 – 55 ). Compared with three other cases of editing phage genes using the CRISPR–Cas9 system, the effect of SpyCas9:RNA on the random mutation selection of phage target sequences and their PAM in this study was similar to that in the study of Lemay ( 43 ), which found almost no point mutations in the target sequence and its PAM among single plaques. However, the subsequent orf6 deletion experiments in this study showed that the editing efficiency was higher than that in the Lemay study and was similar to those in the studies by Tao ( 44 ) and Shen ( 45 ), at approximately 100%.…”

“…However, according to research by Lemay ( 43 ) and Shen ( 45 ) and the results of this study, if the activity of exogenous SpyCas9:RNA is not high and the strain containing the targeted plasmid has a lower effect on the selection of random mutations, then the editing efficiency of the double-plasmid strain for the targeted phage gene depends on the specific situation. For example, the editing efficiency of the nonessential gene orf6 in this study was nearly 100%, and the mutants were all homozygous.…”

Rapid detection of <italic>Escherichia coli</italic> in bloodstream infection via CRISPR-Cas9 engineered reporter phage T7::<italic>Nluc</italic> and microfluidic chip platform

“…In contrast to conventional plaque screening, this method allows the rapid identification of recombinants. Interestingly, we observed heterozygous PCR bands representing both T7 and T7:: Nluc within individual plaques, a phenomenon previously reported in low-activity sgRNA contexts [ 31 , 36 ], but rarely observed with high-affinity sgRNAs. The ELISA approach circumvented this complication, enhancing the overall screening efficiency.…”

Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

“…4d). The phenomenon has also been observed in L. lactis for genome editing of lactococcal phage p2 (16). A possible explanation of those results is that inefficient cleavage can be tolerated through FIG 5 Construction of homologous recombination templates for point mutation, gene deletion, and swap.…”

CRISPR–Cas9 Based Bacteriophage Genome Editing

“…When the exogenous Streptococcus pyogenes SF370 II-A system (CRISPR–Cas9 system) is used to perform gene editing of phage TT4P2 in V. natriegens TT4, it is necessary to determine whether the edited phage harbors anti-Cas9 proteins or an endogenous CRISPR–Cas system, which may cause SpyCas9:RNA inactivity, meaning that it cannot be used as a screening tool ( 53 – 55 ). Compared with three other cases of editing phage genes using the CRISPR–Cas9 system, the effect of SpyCas9:RNA on the random mutation selection of phage target sequences and their PAM in this study was similar to that in the study of Lemay ( 43 ), which found almost no point mutations in the target sequence and its PAM among single plaques. However, the subsequent orf6 deletion experiments in this study showed that the editing efficiency was higher than that in the Lemay study and was similar to those in the studies by Tao ( 44 ) and Shen ( 45 ), at approximately 100%.…”

“…However, according to research by Lemay ( 43 ) and Shen ( 45 ) and the results of this study, if the activity of exogenous SpyCas9:RNA is not high and the strain containing the targeted plasmid has a lower effect on the selection of random mutations, then the editing efficiency of the double-plasmid strain for the targeted phage gene depends on the specific situation. For example, the editing efficiency of the nonessential gene orf6 in this study was nearly 100%, and the mutants were all homozygous.…”

Rapid detection of <italic>Escherichia coli</italic> in bloodstream infection via CRISPR-Cas9 engineered reporter phage T7::<italic>Nluc</italic> and microfluidic chip platform

“…In contrast to conventional plaque screening, this method allows the rapid identification of recombinants. Interestingly, we observed heterozygous PCR bands representing both T7 and T7:: Nluc within individual plaques, a phenomenon previously reported in low-activity sgRNA contexts [ 31 , 36 ], but rarely observed with high-affinity sgRNAs. The ELISA approach circumvented this complication, enhancing the overall screening efficiency.…”

Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

“…4d). The phenomenon has also been observed in L. lactis for genome editing of lactococcal phage p2 (16). A possible explanation of those results is that inefficient cleavage can be tolerated through FIG 5 Construction of homologous recombination templates for point mutation, gene deletion, and swap.…”