Part:BBa_K5179102
dCas9 P4 Cosmid
The dCas9 P4 cosmid is an iteration on the P4 cosmid. Whereas the original P4 cosmid, which was designed by Dr. Fa-Arun (Fa-Arun et al., 2023), was designed to induce sequence specific killing by inducing a double stranded break in a gene target specified by a Cas9 crRNA, the dCas9 cosmid uses a catalytically dead variant of Cas9, meaning one which has abolished DNA cleavage activity but maintained DNA binding activity, to silence target gene expression without killing its bacterial host/chassis (Bikard et al., 2013).
The device includes outward facing BsaI cut sites between the direct repeats of its crRNA region, allowing for easy insertion of a crRNA. The crRNA specifies the target sequence dCas9 will bind to. The crRNA fusion sites are not used in the iGEM type IIS/MoClo assembly standards, thus dCas9 P4 cosmid is suitable for level 0 IIS/MoClo assembly if a crRNA spacer insert with suitable fusion sites is used as a fragment in the assembly.
Usage and Biology
Plasmids containing the dCas9 cosmid can package into the capsids of phage P2, just as the bacteriophage satellite P4 can. In the presence of P2 late genes, P2 terminase will create staggered nicks in the cos site on the device, linearizing any plasmid the device is on, and package it inside a P2 capsid. The device also includes the P4 sid operon which codes for proteins which interface with P2 capsid proteins and cause them to take on an alternative geometry with reduced volume. This effectively excludes large DNA molecules (>30kb) from being packed, such as copies of P2’s chromosome, but permits smaller molecules around the size of P4’s genome (~11.6kb) to pack. P2 structural genes will assemble with the packaged capsids to form mature transducing agents, which can then be used to transduce a plasmid containing this device into new bacterial hosts.
In our project we used the dCas9 P4 cosmid to create transducing units which, upon transduction into their host, silenced a targeted gene. We did this by co-transforming a plasmid carrying the device along with a plasmid expressing the P2 tail fiber protein into E. Coli EMG C5545 ∆cosσε ∆HG (AddGene Bacterial strain #197132), which is lysogenic for a mutant P2 phage which lacks its cos site and tail fiber protein, and has been engineered so that expression of its late genes are expressed by an L-Rhamnose promoter. This bacterial strain, engineered by Dr. Fa-Arun (Fa-Arun et al., 2023), was specially designed for use with the P4 cosmid system. We have shown it to work well with our iterations on the original P4 cosmid and we recommend using it when producing transducing units with this part.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 4825
Illegal NotI site found at 662 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1185
Illegal BamHI site found at 7104
Illegal BamHI site found at 8113
Illegal XhoI site found at 2867 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 8046
Illegal BsaI.rc site found at 8023
Illegal SapI site found at 3095
Characterization
William-and-Mary 2024
To characterize the silencing ability of the dCas9 cosmid we inserted the neomycin phosphotransferase targeting crRNA (BBa_K5179007) into the dCas9 cosmid via a golden gate reaction with BsaI, transformed into E. coli DH5-alpha, harvested the construct and confirmed its sequence with nanopore sequencing.
We produced P4 transducing agent filtrates containing the dCas9 P4 cosmid (with added KanR targeting crRNA)and the original P4 cosmid (Fa-Arun et al., 2023) as a negative control. We treated cultures of E. coli HL713 with each, serially diluted in LB, and plated on LB agar plates with chloramphenicol, kanamycin and chloramphenicol, and no antibiotics. We incubated the plates for 16 hours at 37 C and counted colonies at each dilution level.
Overall treatments with the dCas9 cosmid with a KanR targeting crRNA spacer insert produced a smaller percentage of transductants with resistance to both chloramphenicol and kanamycin, while producing a comparable number of chloramphenicol resistant colonies. First, this indicates that the dCas9 cosmid can be packaged into transducing units as effectively as the original P4 cosmid. Additionally, it suggests some silencing effect on HL713’s kanamycin resistance among transductants. Though this experiment was not sufficiently powered to draw any firm quantitative conclusions about the potency of the dCas9 cosmid’s silencing effect on this target, we wanted to characterize it somewhat, and this result is encouraging enough to motivate further characterization of this device with a different experimental design.
|
Date of Measurement (DD/MM/YYYY) |
dCas9 CFU 10^-5 (Chlor) |
dCas9 CFU 10^-5 (Chlor + Kan) |
Kan resistant colonies/Total transductants (dCas9) |
Original Cosmid (negative control) CFU at 10^-5 (Chlor) |
Original Cosmid (negative control) CFU at 10^-5 (Chlor + Kan) |
Kan resistant colonies/Total transductants (Original Cosmid) |
|
29/09/2024 |
22 |
24 |
1.09 |
32 |
40 |
1.25 |
|
29/09/2024 |
34 |
25 |
0.74 |
27 |
28 |
1.04 |
|
mean |
28 |
24.5 |
0.875 |
29.5 |
34 |
1.15 |
| None |