Construction of pdCas9-w

pdCas9-w contains a gene that produces dCas9 fused to the w subunit of RNA polymerase (RNAP), which recruits RNAP.

Materials and method

• Open pdCas9-bacteria by Phusion PCR (cf. Protocols) with primers f_Gbs_pdCas9 and r_Gbs_pdCas9 + PCR product purification (cf. Protocols) + agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler ladder)
  pdCas9-bacteria was a gift from Stanley Qi (Addgene plasmid # 44249).
• Extract w subunit from pWJ66 by Phusion PCR (cf. Protocols) with primers f_Gbs_w and r_Gbs_w + PCR product purification (cf. Protocols) + agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler)
  pWJ66 was a gift from Luciano Marraffini (Addgene plasmid # 46570).
• Gibson assembly (cf. Protocols) of pdCas9-w using purified PCR products of pdCas9-bacteria and w subunit + transformation (cf. Protocols) of ultra-competent DH5a cells (NEB)
  Control with colony PCR (cf. Protocols) with primers f_Gbs_w and r_Scq_pdCas9_w_sgRNA primers + agarose gel electrophoresis of 2 µL PCR products (with 1kb Generuler) + restriction digest (Cf. Protocols) of pdCas9-w (isolated by Miniprep (cf.Protocols)) with BamHI and KpnI seperately + agarose gel electrophoresis of 2 µL digested products (with 1kb Generuler) + sequencing (Microsynth)
• Site-directed mutagenesis (cf. Protocols) of dCas9-w with primers f_Mt_A2080C_pdCas9-w and r_Mt_A2080C_pdCas9-w (necesary for pdCas9-w to be BioBrick compatible)
  Control with restriction digest (cf. Protocols) of mutated pdCas9-w (isolated by Miniprep (cf. Protocols)) with EcoRI + sequencing (Microsynth)
• BioBrick mutated dCas9-w

Results

Open pdCas9 by PCR

Linearized pdCas9-w is expected to be 6705 bp.
We tested many parameters: HF vs. GC buffer, different annealing temperatures and different extension times. Many, but not all, of our samples were successfully amplified (cf. Fig.1).
For next steps, sample from lane 1 (cf. Fig.1) was used.

Extract w subunit from pWJ66 by PCR

Successful PCR reactions are expected to yield 340 bp fragments.
PCR was succesful for sample visible on gel. (cf. Fig.2)

Gibson assembly of pdCas9-w

Colony PCR

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. It is empty which means there is no contamination.
Gibson assembly seems to have worked for some samples. (cf. Fig.3)
To avoid working with too many samples, we kept the ones from lanes 16, 22, 25, 31, 37 and 43 for the next steps. We did overnight liquid cultures of these colonies.

Restriction digest

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

• Undigested - expected to yield 7 kb circular plasmid (migrates faster than linear fragments of the same size)
• 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
• 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

BamHI and KpnI are both unique cutters in pdCas9 (without the insert) and double cutters in pdCas9-w (with the insert).
Too much ladder was loaded so it is difficult to estimate the size of the fragments. The smaller fragments are also very difficult to see.
By looking at the relative heights, we can say that 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 an error while loading the gel.
Sequencing confirmed that sample 22 is in fact pdCas9-w. We used this sample for the next steps and stored it as a glycerol stock (c.f. Protocols).

Sequencing

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

Construction of pdCas9-w-sgRNAs

These experiments consist of inserting one or two sgRNA producing cassettes (cf. Fig.8a) into pdCas9-w.
pdCas9-w was linearized by restriction digest. The sgRNA cassettes were synthesized (IDT) and necessary overlaps were added by PCR. The sgRNAs were inserted into pdCas9-w by Gibson assembly.

The sgRNA cassettes were synthesized with sequences "A" and "B" at its ends (cf. Fig.8a). These are the sequences those found at the ends of the linearized pdCas9-w.
When one cassette is inserted, no overlaps need to be added.
When two or three cassettes are added, sequences "C" and/or "D" need to be added at the ends of the cassettes (cf. Fig.8b), thus obtaining unique overlaps for the Gibson assembly.

Materials and method

• Open pdCas9-w by restriction digest
  • Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB with Chloramphenicol of pdCas9-w containing bacteria
  • Restriction digest (Cf. Protocols) of pdCas9-w with BsrBI (blunt ends)
  • Agarose gel electrophoresis of 2 µL of digested and undigested product (with 1kb Generuler)
• Amplify sgRNA cassettes by PCR
  • 20 µL Phusion or Q5 PCR (cf. Protocols) of sgRNA cassette with primers as indicated in table below (->Results->Amplify sgRNA cassettes by PCR)
  • Agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler)
  • PCR product purification (cf. Protocols)
• Assemble pdCas9-w-sgRNA constructs by Gibson assembly
  • Part 1: pdCas9-w + sgRNA assembly
    • Gibson assembly (cf. Protocols) with linearized pdCas9-w and purified sgRNA PCR products:
    • 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 primers f_ColPCR_sgRNAs and r_ColPCR_sgRNAs primers + agarose gel electrophoresis of 2 µL PCR prodcuts (with 1kb Generuler)
      One colony PCR (cf. Fig11a) was done with primers f_Gbs_pdCas9 and r_Scq_pdCas9_w_sgRNA
    • Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB with Chloramphenicol of colonies from plate used for culture of transformed cells to isolate pdCas9-w plasmids
    • Sequencing (Microsynth) of one colony for which Gibson assembly worked according to colony PCR and restriction analysis
  • Part 2: pdCas9-w-sgRNA + sgRNA assembly

Results

Open pdCas9-w by restriction digest

The digested and undigested plasmids should have the same size. Circular fragments migrates faster than linear fragments of the same size. This means that the undigested plasmid should migrate faster than the digested plasmid.
Even though the ladder is not clear, by looking at the relative heights we can see that the digested sample migrated more slowly than the undigested sample (cf. Fig.9). The restriction digest was successful.

Note that this was repeated several times since we needed a lot of linearized pdCas9-w.

Amplify sgRNA cassettes by PCR

sgRNA cassette(s)	Forward primer(s) / Reverse primer(s)
Z0	f_Gbs_sgRNA-A / r_Gbs_sgRNA-B f_Gbs_sgRNA-A / r_Gbs_sgRNA-BtoD
Z4	f_Gbs_sgRNA-A / r_Gbs_sgRNA-B
Z35	f_Gbs_sgRNA-A / r_Gbs_sgRNA-B
X0	f_Gbs_sgRNA-A / r_Gbs_sgRNA-B
X4	f_Gbs_sgRNA-A / r_Gbs_sgRNA-B
X35	f_Gbs_sgRNA-A / r_Gbs_sgRNA-B

All amplified sgRNA cassettes are expected to be about 370 bp.

sgRNA cassette Z0 was amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-BtoD and primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Q5 PCR. Both samples were successfully amplified (cf. Fig.10a).

sgRNA cassette Z4 was amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. Samples 3 and 4 were successfuly amplified (cf. Fig.10b).

sgRNA cassette Z35 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. However, it is not easily visible on the gel. (cf. Fig.10c)

sgRNA cassette X0 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10d)

sgRNA cassette X4 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10e)

sgRNA cassette X35 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10f)

Assemble pdCas9-w-sgRNA constructs by Gibson assembly

Part 1: pdCas9-w + sgRNA assembly

In colony PCR, primers were placed such as amplicons are 445 bp if the plasmid self-ligated, 723 bp if Gibson assembly of 1 sgRNA cassette worked and 1120 bp if Gibson assembly of 2 sgRNA cassettes worked.

pdCas9-w-Z0 was obtained by a faulty assembly of pdCas9-w-Z0-Z4 that only took up 1 of the 2 sgRNA cassettes. Sample 20 of the colony PCR (cf. Fig.11a) was confirmed by sequencing to be pdCas9-w-Z0 and to not contain any mutations that may have an effect on its activity. Sample 13 seems to be pdCas9-w-Z0-Z4 according to colony PCR (cf. Fig.11a), but sequencing showed that it is pdCas9-w-X0-Z0, probably due to contamination of a tube. (This sample will be used again in Part 2.)
Colony PCR showed that all colonies tested for pdCas9-w-Z4 have the inserted sgRNA cassette (cf. Fig.11b). Sequencing comfirmed that sample 5 is in fact pdCas9-w-Z4. Colony PCR showed that the Gibson assembly for sample 5 of pdCas9-w-Z35 seems to have worked (cf. Fig.11c) and sequencing confirmed that is the case. Sequencing also showed some mutations or deletions in the promoter and/or terminator for these 2 samples. We decided to keep working with these and test whether these mutations are significative with an activity assay.
Colony PCR and sequencing showed that Gibson assembly was successful for sample 5 of pdCas9-w-X0 (cf. Fig.11d). However, it contains a mutation in the promoter. A 2nd pdCas9-w-X0 without mutations will be constructed in part 2, after which we will be able to compare the activity of this promoter with/without a mutation.
Colony PCR of pdCas9-w-X4 showed many potentially good samples (cf. Fig.11e). Sequencing of sample 10 confirmed that it is in fact pdCas9-w-X4 and that it does not contain any mutations.
Colony PCR and sequencing showed that Gibson assembly was successful for the colony of pdCas9-w-X35 (cf. Fig.11d) and no mutations were found in the sequence.

Part 2: pdCas9-w-sgRNA + sgRNA assembly

Construction of pWJ89alt

pWJ89alt contains GFP controlled by the promoter J23117alt, which can be activated by dCas9-w bound to sgRNA X4 and inhibited by dCas9-w bound to sgRNA X0 or X35.

Materials and method

• PCR pWJ89 wihtout its J23117 promoter. pWJ89 was a gift from David Bikard.
  • Miniprep (cf. Protocols) liquid culture of bacteria containing pWJ89
  • 20 µL Phusion PCR and 25 µL Q5 PCR (cf. Protocols) of pWJ89 using primers f_Rmv_J23117_of_pWJ89 and r_Rmv_J23117_of_pWJ89.
  • Agarose gel electrophoresis of 2 µL PCR products (with 1kb Generuler)
  • PCR product purification (cf. Protocols)
• PCR J23117alt promoter (synthsized by IDT)
  • 20 µL Phusion PCR and 25 µL Q5 PCR (cf. Protocols) of J23117alt using primers f_G_J23117Alt1IDT and r_G_J23117Alt1IDT.
  • Agarose gel electrophoresis of 2 µL PCR products (with 1kb Generuler)
  • PCR product purification (cf. Protocols)
• Gibson assembly of pWJ89alt
  • Gibson assembly (cf. Protocols) with purified PCR products pWJ89 (without J23117) and J23117alt promoter
  • Transformation (cf. Protocols) of ultra-competent DH5a cells (NEB) with Gibson assembly product, spreading on Kanamycin plates
  • Colony PCR (cf. Protocols) of colonies from plate used for culture of transformed cells with primers f_Cl_pWJ89 and r_Sq_J23117alt primers + agarose gel electrophoresis of 2 µL PCR prodcuts (with 1kb Generuler)
  • Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB with Kanamycin and sequencing (Microsynth) of one colony for which Gibson assembly worked according to colony PCR
• BioBrick J23117alt promoter

Results

PCR pWJ89

Successful PCR reactions are expected to yield 4400 bp amplicons.
Both negative control lanes (cf. Fig.13) are empty, which means there is no contamination. Phusion PCR did not work, but Q5 PCR did (cf. Fig.13). We will work with this sample for next steps.

PCR J23117alt promoter

Successful PCR reactions are expected to yield 340 bp amplicons.
Both negative control lanes (cf. Fig.14) are empty, which means there is no contamination. Both Phusion and Q5 PCR worked (cf. Fig.13). We will work with the sample amplified by Q5 PCR.

Construction of pWJ89alt_Z4-to-X4

pWJ89alt_Z4-to-X4 contains a cassette producing sgRNA X4 (activator for J23117alt) controlled by the promoter J23117 and GFP under the J23117alt promoter (corresponds to pWJ89alt).

Materials and method

• Open pWJ89alt by restriction digest + Agarose gel electrophoresis of 2 µL of digested product (with 1kb Generuler)
• It was constructed from pWJ89alt (as described above) and Z4-to-X4, the cassette producing sgRNA X4 controlled by the promoter J23117

  pWJ89alt was linearized by restriction digest by AfeI, Z4-to-X4 was amplified by PCR and both were assembled by Gibson assembly.

  Open pWJ89alt by restriction digest

  Coming soon

  PCR Z4-to-X4

  Coming soon

  Gibson assembly pWJ89alt_Z4-to-X4

  Coming soon

Construction of pWJ89_mCherry

Coming soon

Transistor activity assay

Coming soon

Primer table