Production of P1 phage with SaCas9/FnCpf1
Three kinds of P1 phages with SaCas9/FnCpf1 were produced: one carrying the VanA spacer, one with the blaKPC spacer, and one without any spacer (negative control).
Strategy to check the efficiency of the CRISPR system and phage infection
To check the efficiency of the CRISPR system and phage infection, we took advantage of different antibiotic resistant genes (Figure 1). The MoClo cells possess the target sequence of the CRISPR system on a KanR plasmid. On the contrary, the SaCas9/FnCpf1 system is on the ChlrR P1 phagemid. Therefore, if cleavage by the endonucleases occurs, it results in the loss of Kanamycin resistance gene in the host cell, while in the acquisition of Chloramphenicol resistance. In both scenarios in which the endonucleases work or don’t work, the MoClo cells will possess ChlrR as the result of the phage infection.
To test it, from the Kanamycin and Chloramphenicol plates (Figure 1 Step 1), several colonies were picked, and each colony was directly inoculated onto new LB plates (with Chloramphenicol, Kanamycin, and no antibiotics) (Figure 1 Step 2). The efficiency of the CRISPR system was verified by the presence of colonies when the colonies were moved from a Chloramphenicol plate to a Kanamycin plate. If these colonies do not grow on Kanamycin plates, we can assess that the cleavage of the target sequence by endonucleases occurred, leading to the loss of the KanR MoClo plasmid. Instead, the efficiency of the phage infection was assessed by counting the colonies on the Chlr plate.
Figure 1: Strategy to verify the efficiency of the SaCas9 system and phage infection.
Efficiency of SaCas9 system
To investigate the efficiency of the SaCas9 system to cleave the target sequence, colonies were picked from Chloramphenicol plates and inoculated onto Kanamycin plates (Figure 1 Step 2). Colonies 1-4 came from KPC MoClo cells infected by P1-SaCas9-KPC spacer, while colonies 5-8 were VanA MoClo cells infected with P1-SaCas9-vanA spacer. In addition, colonies from KPC and VanA MoClo cells infected by the negative control phage (no spacers) were also picked (A-D for KPC MoClo cells, E-H for VanA MoClo cells). The result is shown in Table 1. The pictures of plates are available in Figure 2.
The SaCas9 system targeting the KanR plasmid within VanA MoClo cells showed promising - albeit not consistent - results. Among colonies 5-8 from the Chloramphenicol plate, colony 6 did not grow on the Kanamycin plate indicating that the SaCas9 might have cleaved the target KanR plasmid. However, the experiment was performed only once and further replicates to confirm the efficiency of the SaCas9 should be tested.
On the contrary, survival of the KPC MoClo cells on the Kanamycin plates implied that none of the plasmid with blakpc target sequences and the Kanamycin resistance gene were cleaved. This may be due to an error in the spacer design, as SaCas9 cassettes are identical between ones targeting blaKPC and ones for VanA.
Despite these results, three colonies which was infected by negative control phage did not grow on Kanamycin plates. This might imply that KanR plasmid has been lost from the MoClo cells due to unknown mechanism. Further repetitive experiments are needed, to make sure that our SaCas9 is functional.
Efficiency of phage infection
The efficiency of phage infection suggested to be low (Table 1). This is because the colonies 9-16 from Kanamycin plates did not grow on Chloramphenicol plates, thus not hindering the P1 phagemid. Quantitative analysis on the number of phage in solution could have been done for more precise results.
Table 1: Verification of efficiency of the CRISPR SaCas9 system and phage infection. Vertical axis shows original plates. Horizontal axis shows the new plates which were inoculated colonies from the original plate.
Figure 2: Verification of efficiencies of the SaCas9 system and phage infection.
Verification of efficiency of the FnCpf1 system and phage infection
P1-FnCpf1 seems to have cleaved the target sequence in colony No. 16 and 19 (Table 2A, Figure 3). However, P1-FnCpf1 targeting blaKPC failed to cleave any target sequence.
In addition, the efficiency of phage infection appeared to be low, because only one out of 12 colonies from Kanamycin plates could survive on Chloramphenicol plates.
Table 2A: The efficiency of FnCpf1 system.
Table 2B: Classification of colonies.
Status1: engineered phage infected Moclo cells successfully because they obtained Chl resistance from Phagemid, but CRISPR did not work.
Status2 :phage did not infect Moclo successfully because phagemid was not injected into.
Status3 :engineered phage infected Moclo successfully and CRISPR did work.
Status4: they cannot grow even on Kan plates due to unkown reason.
Figure 3: P1-FnCpf1 seems to have cleaved the target sequence in colony No. 16 and 19.
Colonies 16 and 19 are VanA MoClo cells infected by P1-FnCpf1-vanA spacer. They grew on LB and Chloramphenicol plate, but not Kanamycin plate. This pattern of growth suggests the cleavage of target KanR plasmid in MoClo cells. Even though there is bacteria growing on Kanamycin plate in the area for colony 19, this is leakage from colony F.
F and K are MoClo cells infected by negative control phage (no spacer). They grew on all three plates.