In a previous checkoﬀ‐funded study (Yang et al., 2017), proof‐of‐concept work demonstrated that the CRISPR‐ Cas9 system with guide RNA (gRNA) could selectively kill Shiga toxin‐producing Escherichia coli (STEC) cells by targeting their Shiga toxin genes, stx1 and/or stx2. Furthermore, the killing eﬃciency of the CRISPR‐Cas9 system against STEC cells was improved by optimizing the design of, and using multiple, gRNAs. Speciﬁcally, researchers constructed a pCRISPR with two gRNAs that can selectively kill STEC cells containing the stx1 and/or stx2 genes. Because of the simultaneous cleavage of the chromosome at two (rather than one) locations, this pCRISPR cloned with two gRNAs achieved signiﬁcantly greater reductions of E. coli O157:H7 cells than pCRISPRs cloned with only a single gRNA.
The CRISPER‐Cas9 mediated killing system changes the current understanding about how to reduce or eliminate pathogen contamination on meat products. This is important for the meat industry because the CRISPR‐Cas9 system could serve as a novel antimicrobial intervention for the control of foodborne pathogens. For example, gRNAs can be designed to target (i) virulence genes for sequence‐speciﬁc removal of foodborne pathogens other than STEC (e.g., Salmonella and Listeria monocytogenes), (ii) antibiotic resistance genes for killing antibiotic‐resistant bacteria, and (iii) genes involved in bioﬁlm development and formation of bacterial persister cells in bioﬁlms to prevent bioﬁlm formation, and to improve sanitizer eﬃciency against bioﬁlms in meat processing environments.
Currently, the main obstacle for the application of this technology in meat production and processing environments is an eﬃcient delivery mechanism for the CRISPR‐Cas9 system into bacterial cells. Recently, use of bacteriophage has been shown to be a promising delivery vehicle for introducing CRISPR‐Cas9 antimicrobials into living cells (Luo et al., 2016; de la Fuente‐Núñez et al., 2017). For example, Bikard et al. (2014) used a phage system to deliver the Cas9 gene and its gRNA into Staphylococcus aureus cells for sequence‐speciﬁc killing of kanamycin‐resistant cells.
Yosef et al. (2015) engineered a prophage to deliver the type I‐E CRISPR‐Cas system targeting plasmid‐borne ß‐lactamase genes to sensitize and kill antibiotic‐resistant bacteria. In this study, researchers focused on developing a phage‐ mediated delivery system that would allow the delivery of the CRISPR‐Cas9‐based antimicrobials into target STEC cells.
The overall goal of the project was to construct a phage‐mediated system for the efficient delivery of the CRISPR‐ Cas9 antimicrobials into bacterial cells, and the specific objectives were to:
- Synthesize a DNA fragment with sequences encoding Cas9 proteins and guide RNAs that target Shiga toxin 1 and 2 genes.
- Clone this DNA fragment into a phagemid vector so that the resulting phagemid vector can successfully express Cas9 proteins and the guide RNAs.
- Package the phagemid with the CRISPR‐Cas9 system targeting Shiga toxin 1 and 2 genes into a helper phage.
- Infect E. coli O157:H7 cells with the phage stock that carries the CRISPR‐Cas9 system targeting Shiga toxin 1 and 2 genes and evaluate kill efficiency.
To develop a phagemid system suitable for E. coli O157:H7, a ~5‐kb fragment that contains tracrRNA, Cas9, crRNA and two 20‐bp spacers (one spacer targeting the stx1 gene and the other spacer targeting the stx2 gene) was synthesized. The synthesized fragment was mainly based on the genomic sequence of Streptococcus pyogenes M1GAS with some modiﬁcations (Bikard et al., 2014) (Figure 1). The synthesized fragment was cloned into the phagemid vector, pBluescript KS (+) (Agilent Technologies, Santa Clara, CA), to construct a phagemid that carries the CRISPR‐Cas9 system targeting the stx1 and stx2 genes (Figure 2). A control phagemid that carried tracrRNA, Cas9, and crRNA but without any spacers was also constructed (Figure 2).
The constructed phagemids were separately introduced into four E. coli O157:H7 strains to evaluate their cell reductions. These four E. coli O157:H7 strains had diﬀerent Shiga toxin gene proﬁles: the Sakai strain has both the stx1 and stx2 genes; the C1‐158 strain has only the stx1 gene; the C1‐010 strain has only the stx2 gene, and the C1‐057 strain has neither the stx1 nor the stx2 gene. Phagemids that carried the CRISPR‐Cas9 system targeting the stx1 and stx2 genes were packaged into M13KO7 helper phages (Figure 4). Additionally, control phagemids without any gRNAs were packaged into M13KO7 phages as controls (Figure 4).
Other than the pure culture of E. coli O157:H7 in 2‐YT broth, the eﬃcacy of the antimicrobial CRISPR‐Cas9 system was also evaluated in an ex vivo model rumen system. Fresh cattle rumen ﬂuid containing 107 CFU/reaction of natural microﬂora was inoculated with E. coli O157:H7 (F’) cells at the concentration of 106 CFU/reaction. The inoculated fresh cattle rumen ﬂuid was infected with the M13KO7 helper phages packaged with the CRISPR‐Cas9 system targeting the stx1 and stx2 genes.
Introduction of the phagemids into the stx1‐ and/or stx2‐containing E. coli O157:H7 strains resulted in signiﬁcant (P < 0.05) cell reductions and the reductions were in the order of: Sakai > C1‐158 = C1‐010 (Figure 3). No signiﬁcant (P > 0.05) reductions were observed for the C1‐057 strain, which lacked both Shiga toxin genes (Figure 3). The signiﬁcantly higher reductions of E. coli O157:H7 cells containing both the stx1 and stx2 genes could be explained by the
simultaneous cleavage of the chromosome at two locations rather than just one location.
Overall, the results obtained for the E. coli O157:H7 strains with diﬀerent Shiga toxin gene proﬁles indicated that the constructed phagemid is highly speciﬁc to the stx1 and stx2 genes. The constructed phagemid that carries the CRISPR‐Cas9 antimicrobials will only eﬃciently kill Shiga toxin‐producing Escherichia coli (STEC) cells by speciﬁc recognition and cleavage of the bacterial chromosome at the sites of the stx1 and stx2 genes, not anywhere else.
A signiﬁcant (P < 0.05) reduction of E. coli O157:H7 cells, from 6.96±0.10 to 4.44±0.10 log CFU/reaction, was achieved at a MOI of 25 (Table 2). At a MOI of 0.25, a signiﬁcant (P < 0.05) reduction of E. coli O157:H7 cells, from 5.44±0.12 to 2.70±0.12 log CFU/reaction, was achieved as well (Table 1). These results suggested that the complex microbial communities present in cattle rumen ﬂuid did not interfere with the CRISPR‐Cas9 antimicrobial eﬃcacy of the developed phages. The kill eﬃciencies were well retained when the phages that carry the CRISPR‐Cas9 antimicrobials were used to reduce E. coli O157:H7 cells inoculated in cattle rumen ﬂuid. Therefore, it is promising that the developed phages could be applied as a means of reducing the carriage of STEC cells in ruminant animals.
The CRISPR‐Cas9‐based antimicrobial system constructed in the current work will potentially be applied in food animal production environments. Currently, the biosafety aspects of the CRISPR‐based technique are still under international debate. To prepare for the potential biosafety concerns that may arise when applying the CRISPR‐ Cas9 antimicrobials to food animals, the design of the gRNAs was modiﬁed by extending the length of the spacers to avoid oﬀ‐target eﬀects. In addition to phagemids containing two 20‐bp spacers, new phagemids containing two 60‐bp spacers targeting the same stx1 and stx2 gene areas were constructed. Phagemids containing two 60‐bp spacers killed signiﬁcantly more (P < 0.05) E. coli O157:H7 cells (from 4.21±0.13 to 0.50±0.13 log CFU/reaction) than phagemids containing two 20‐bp spacers (from 4.21±0.13 to 1.86±0.13 log CFU/reaction) when they were introduced into E. coli O157:H7 cells.
The development of phages that can successfully deliver the CRISPR‐Cas9‐mediated antimicrobials into bacterial cells oﬀers a solution to overcome the ﬁrst obstacle incurred during the application of the CRISPR‐Cas9 technique in actual meat production and processing environments. The developed CRISPR‐Cas9‐carrying phages could potentially serve as a novel antimicrobial intervention and/or an alternative to antibiotics for selective killing of bacterial pathogens in food animals in the pre‐harvest environment. Results from the study indicate that the complex microbial community present in cattle rumen ﬂuid will not interfere with the antimicrobial eﬃcacy of the developed phages. Furthermore, this research explored the strategy of designing longer gRNA to achieve enhanced speciﬁcity and eﬃcacy. This approach oﬀers another opportunity to overcome a second potential obstacle, i.e. biosafety concerns, that may be encountered when applying the CRISPR‐Cas9 technique in vivo in the future.