Assemble pdCas9-w: Open pdCas9 by PCR

We received plasmid pdCas9 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.
pdCas9 is a plasmid containing dCas9 under a Tetracyclin inducible promoter and a Chloramphenicol resistance gene, available on Addgene under the name pdCas9-bacteria.
We opened this plasmid by PCR to be able use later on in a Gibson assembly.

Materials and method

• 20 µl Phusion PCR (cf. Protocols) of 1 ng pdCas9 with appropriate primers, testing parameters such as HF vs. GC buffer, annealing temperatures and extension times
• PCR product purification (cf. Protocols)
• Agarose gel electrophoresis of purified PCR products

Results

Linearized pdCas9-w is expected to be 6705 bp.
After testing many parameters, we were able to linearize the plasmid succesfully, as seen on gel above.
For further uses, sample from lane 1 was used. It was prepared with HF buffer and the following thermocycling settings:

Assemble pdCas9-w: Extract w subunit from pWJ66 by PCR

We received plasmid pWJ66 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.
pWJ66 is a plasmid containing dCas9 fused at its C-terminal to the w subunit of RNA polymerase as well as a tracrRNA gene and a CRISPR array gene, available on Addgene.
We extracted the w subunit to fuse it to our own dCas9 by Gibson assembly.

Materials and method

• 20 µl Phusion PCR (cf. Protocols) with 1 ng pWJ66 using appropraite primers, HF buffer and following thermocycling settings:

Step		Temperature	Time
Initial Denaturation		98°C	30 seconds
30 cycles	Denaturation	98°C	10 seconds
	Annealing	62°C	15 seconds
	Extension	72°C	15 seconds
Final Extension		72°C	7 minutes
Hold		4°C

• PCR product purification (cf. Protocols)
• Agarose gel electrophoresis of purified PCR products

Results

Successful PCR reactions are expected to yield 340 bp fragments.
As seen on gel, PCR was succesful for sample in lane 1.

Assemble pdCas9-w: Gibson assembly of pdCas9-w

Using PCR products from experiments described above, we fused the w subunit of DNA polymerase to dCas9 by Gibson assembly.

Materials and method

• Gibson assembly (cf. Protocols) with purified PCR products:
• Open pdCas9: 0.02 pmol = 95 ng
• w subunit extracted from pWJ66: 0.06 pmol = 12.5 ng
• Transformation (cf. Protocols) of ultra-competent DH5a cells (NEB) with Gibson assembly product, spreading on Chloramphenicol plates
• Colony PCR (cf. Protocols) of colonies from plate used for culture of transformed cells with appropriate primers and following thermocycling setttings:

Step		Temperature	Time
Initial Denaturation		95°C	30 seconds
30 cycles	Denaturation	98°C	30 seconds
	Annealing	56°C	35 seconds
	Extension	68°C	80 seconds
Final Extension		68°C	5 minutes
Hold		4°C

• Isolate pdCas9-w plasmids by Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB + Chloramphenicol of colonies from plate used for culture of transformed cells
• Restriction digest (Cf. Protocols) of pdCas9-w plasmids with BamHI and KpnI seperately, both are unique cutters in pdCas9 (without the insert) and double cutters in pdCas9-w (with the insert)
• Sequencing (Microsynth) of one colony for which Gibson assembly worked according to colony PCR and restriction analysis

Results

Gibson assembly and transformation

Almost as many colonies grew in our sample as in the self-ligation control (Gibson protocol with pdCas9 but no w subunit). This may mean that most of the colonies in our sample are actually self-ligation and that the Gibson assembly did not work.
However, the transformation control (pUC19, NEB) worked very well.

Colony PCR

Primers were placed such as amplicons are 666 bp if Gibson assembly worked and 396 bp if the plasmid self-ligated.
Lane "C" is a negative control, PCR was run with all components except template DNA, ie. cells. It is empty, this means there is no contamination.
As we can see on the gels, Gibson assembly may have worked for samples in lanes 5, 6, 16, 22, 25, 26, 27, 28, 29, 30, 31, 37, 38, 40, 41 and 44.
To avoid working with too many samples, we kept the ones from lanes 16, 22, 25, 31, 37 and 43 for the Minipreps and restriction digest. We did an overnight liquid culture of the corresponding colonies.

Restriction digest

pdCas9-w samples from different colonies are present on gels in triplicates in the following order:

1. Undigested: expected to yield 7 kb circular plasmid (migrates faster than linear fragments of the same size)
2. Digested by BamHI: expected to yield two fragments of 6147 bp and 834 bp if insert is present or one 6705 bp fragment if it is not
3. Digested by KpnI: expected to yield two fragments of 45334 bp and 2447 bp if insert is present or one 6705 bp fragment if it is not

As visible on the gels, all colonies seem to have the w subunit insert. There is only the undigested sample for colony 16 that is not visible, probably due to not loading the gel correctly.
Colony 22 was kept for next steps, it was stored in a glycerol stock (c.f. Protocols).

Sequencing

As dCas9-w is very long, only part of it (the w subunit and its surrounding base pairs) was sequenced and no mutations were detected.