02. May
PCR of pX334 fragments
PCR 1
| µl |
type |
| 10 |
Q5-HF Reaction Buffer |
| 1 |
pX334 |
| 1 |
oIG2000 |
| 1 |
oIG2029 |
| 2.5 |
dNTPs |
| 1.5 |
DMSO |
| 0.5 |
Q5-HF Polymerase |
| Add to 50 |
H2O |
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 2
| µl |
type |
| 10 |
Q5-HF Reaction Buffer |
| 1 |
pX334 |
| 1 |
oIG2028 |
| 1 |
oIG2001 |
| 2.5 |
dNTPs |
| 1.5 |
DMSO |
| 0.5 |
Q5-HF Polymerase |
| Add to 50 |
H2O |
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 3
| µl |
type |
| 10 |
Q5-HF Reaction Buffer |
| 1 |
pX334 |
| 1 |
oIG2002 |
| 1 |
oIG2003 |
| 2.5 |
dNTPs |
| 1.5 |
DMSO |
| 0.5 |
Q5-HF Polymerase |
| Add to 50 |
H2O |
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 4
| µl |
type |
| 10 |
Q5-HF Reaction Buffer |
| 1 |
pX334 |
| 1 |
oIG2006 |
| 1 |
oIG2007 |
| 2.5 |
dNTPs |
| 1.5 |
DMSO |
| 0.5 |
Q5-HF Polymerase |
| Add to 50 |
H2O |
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
PCR 5
| µl |
type |
| 10 |
Q5-HF Reaction Buffer |
| 1 |
pX334 |
| 1 |
oIG2008 |
| 1 |
oIG2009 |
| 2.5 |
dNTPs |
| 1.5 |
DMSO |
| 0.5 |
Q5-HF Polymerase |
| Add to 50 |
H2O |
- Annealing: 60°C
- Extension: 115 sec.
- Cycles: 18
All samples were stored at -20°C
02. May
Agarose Gelelectrophoreses of PCR fragments No. 1-5
 |
| f.l.t.r.: marker, PCR 1 (1909 bp), PCR 2 (2610 bp), PCR 3 (1645 bp), PCR 4 (2272 bp), PCR 5 (1878 bp) |
Gel Extraction of PCRs 1-6
| name |
ng/µl |
| Fragment No. 1 |
40.7 |
| Fragment No. 2 |
53.6 |
| Fragment No. 3 |
52.6 |
| Fragment No. 4 |
41.6 |
| Fragment No. 5 |
40.4 |
03. May
Fusion PCR of PCR fragments 2-3 and 5-1
Assembly fragment 2-3 (PCR 1)
| µl |
type |
| 10 |
Q5-HF Reaction Buffer |
| 1 |
Fragment No. 2 |
| 1 |
Fragment No. 3 |
| 4 |
dNTPs |
| 1.5 |
DMSO |
| 0.5 |
Q5-HF Polymerase |
| Add to 50 |
H2O |
- Annealing: 71°C
- Extension: 105 sec.
- Cycles: 5
Assembly fragment 2-3 (PCR 2)
- Addition of 1 µl of oIG2028 and oIG2003 to PCR 1
- Annealing: 60 °C
- Extension: 2 min 40 sec
- Cycles: 16
Assembly fragment 5-1 (PCR 1)
| µl |
type |
| 10 |
Q5-HF Reaction Buffer |
| 1 |
Fragment No. 5 |
| 1 |
Fragment No. 1 |
| 4 |
dNTPs |
| 1.5 |
DMSO |
| 0.5 |
Q5-HF Polymerase |
| Add to 50 |
H2O |
- Annealing: 71°C
- Extension: 105 sec.
- Cycles: 5
Assembly fragment 5-1 (PCR 2)
- Addition of 1 µl of oIG2008 and oIG2029 to PCR 1
- Annealing: 60 °C
- Extension: 2 min 40 sec
- Cycles: 16
Gel Extraction of Fusion fragments 2-3 and 5-1
| name |
ng/µl |
| Fragment No. 2-3 (a) |
76.6 |
| Fragment No. 2-3 (b) |
57.5 |
| Fragment No. 2-3 (c) |
106.8 |
| Fragment No. 5-1 |
23.4 |
17. May
PCR of the KRAB fragment with a N-terminal linker (GSAGSAG) overhang
PCR 1
| µl |
type |
| 10 |
Q5-HF Reaction Buffer |
| 1 |
pKM102 |
| 1 |
oIG2004 |
| 1 |
oIG2005 |
| 2.5 |
dNTPs |
| 1.5 |
DMSO |
| 0.5 |
Q5-HF Polymerase |
| Add to 50 |
H2O |
- Annealing: 60°C
- Extension: 30 sec.
- Cycles: 18
- For higher Gel Extraction yields: 4x amount
Agarose Gelelectrophoreses of the KRAB fragment
 |
| f.l.t.r.: marker, KRAB at 490 bp (4x) |
Gel Extraction of the KRAB fragment
| name |
ng/µl |
| KRAB (a) |
76.0 |
| KRAB (b) |
64.7 |
Two bands were respectively pooled on one extraction column, in order to yield higher amount of DNA.
20. May
Gibson Assembly of pIG2004 (Cas9-KRAB)
- Preparation of 5µl DNA mix, containing all four fragments
- Melting a 15µl master-mix on ice, until it is ready to use
- Addition of 5µl of DNA mix to the master mix
- Immediately put the mix on 50°C and incubate for 1h
- 3 min on RT
- 3 min on ice
- Transformation of 4 µl to TOP10 chemically competent E-coli cells
 |
| Preparation scheme for Gibson Assembly fragment mix |
21. May
Evaluation of Gibson Assembly
- Colony growth
- Colony PCR will be performed with primers within KRAB and Cas9 fragment
Colony PCR Mastermix for Gibson Clones
| µl |
type |
| 34.8 |
H2O |
| 13.0 |
Taq Buffer |
| 13.0 |
dNTPs |
| 1.3 |
oIG2002 |
| 1.3 |
oIG2005 |
| 1.6 |
Taq Polymerase |
| 5.0 |
H2O for each reaction, containing picked colony |
- Annealing: 60°C
- Extension: 68°C
- Extension: 2 min 10 sec.
- Cycles: 30
 |
| Results of Colony PCR: Clones 5, 6 and 7 were striked out. |
22. May
Miniprep of suspected pIG2004 Clones 5, 6 and 7
| name |
ng/µl |
| pIG2004, No. 5 |
207.9 |
| pIG2004, No. 6 |
180.0 |
| pIG2004, No. 7 |
198.1 |
Clones were sent to sequencing at GATC
23. May
Evaluation of Gibson Cloning Sequencing Results
 |
| oIG0007 sequencing reveals that the KRAB fragment was succesfully fused to Cas9 in each case. Further sequecing will be done to test for correct N-terminal assembly and H840A mutation. |
PCR of pX334 fragment 6
PCR 1
| µl |
type |
| 10 |
Q5-HF Reaction Buffer |
| 1 |
pX334 |
| 1 |
oIG2002 |
| 1 |
oIG2007 |
| 2.5 |
dNTPs |
| 1.5 |
DMSO |
| 0.5 |
Q5-HF Polymerase |
| Add to 50 |
H2O |
- Annealing: 60°C
- Extension: 2 min 40 sec.
- Cycles: 18
Agarose Gelelectrophoreses of PCR fragments No. 6
 |
| f.l.t.r.: marker, PCR 6 (3908 bp), empty, PCR 6 (2x) |
Gel Extraction of PCR 6
| name |
ng/µl |
| Fragment No. 6 |
43.6 |
Gibson Assembly of pIG2005 (dCas9)
- Preparation of 5µl DNA mix, containing all four fragments
- Melting a 15µl master-mix on ice, until it is ready to use
- Addition of 5µl of DNA mix to the master mix
- Immediately put the mix on 50°C and incubate for 1h
- 3 min on RT
- 3 min on ice
- Transformation of 4 µl to TOP10 chemically competent E-coli cells
 |
| Preparation scheme for Gibson Assembly fragment mix |
24. May
Digest of pIG2004 (No. 5, 6 and 7) with BbsI
| µl |
type |
| Volume |
DNA |
| 16 |
pIG2004 |
| 10 |
NEB-Buffer 2.1 |
| 2 |
BbsI |
| 72 |
H2O |
- Temp.: 37°C
- Incubation time: 2h
Agarose Gelelectrophoreses of pIG2004 Digests
 |
| f.l.t.r.: marker, pX334, empty, digested pIG2004 5 (2x), 6 (2x) and 7 (2x). Upper bands of clone no. 5 and 6 digests were cut out for Gel Extraction. |
Gel Extraction of pIG2004 digest
| name |
ng/µl |
| pIG2004 (5) |
38.0 |
| pIG2004 (6) |
17.0 |
Evaluation of Gibson Assembly of 23. May
- Colony growth
- Five clones were striked out
27. May
Evaluation of pIG2004 sequencing results (No. 5 and 6)
- H840A mutation was inserted in both cases, leading to catalytic inactivation of Cas9
- a 300 bp gap was inserted within the CAG promoter, probably due to error-prone PCR amplification
 |
| Very GC-rich part on pIG2004 template (part of the CAG promoter lacks nearly 300 bp after having been amplified via PCR and subsequently Gibson-assembled. |
04. June
Minipreps of pIG2005 clones striked out on 23. May
| name |
ng/µl |
| pIG2005 - No. 1 |
77.1 |
| pIG2005 - No. 2 |
68.4 |
| pIG2005 - No. 3 |
59.6 |
| pIG2005 - No. 4 |
43.7 |
| pIG2005 - No. 5 |
61.2 |
07. June
Fixing of the 300 bp CAG promoter gap for Cas9-KRAB
Restriction digest of pIG2004 and pIG2005 - 1
| µl |
type |
| 8 |
pIG2004 - No. 5 or pIG2005 - No. 1 |
| 8 |
NEB-Buffer 4 |
| 2 |
AgeI-HF |
| 2 |
NotI-HF |
| 2 |
SacII |
| 2 |
BSA |
| Add to 80µl |
H2O |
Restriction digest of pX334 - 2
| µl |
type |
| 8 |
pX334 DNA |
| 8 |
NEB-Buffer 4 |
| 2 |
KpnI-HF |
| 2 |
NotI-HF |
| 2 |
SacI-HF |
| 2 |
BSA |
| Add to 80µl |
H2O |
Restriction digest of pX334 - 3
| µl |
type |
| 8 |
pX334 DNA |
| 8 |
NEB-Buffer 4 |
| 2 |
KpnI-HF |
| 2 |
AgeI-HF |
| 2 |
BSA |
| Add to 80µl |
H2O |
- Temp.: 37°C
- Incubation time: 1h
- Expected fragment lengths: 1 - 4500 bp (Cas9-KRAB), 2 - 4900 bp (Backbone), 3 - 890 bp (CAG promoter)
 |
| Top left. Upper band: Cas9-KRAB fragment, Top right. Upper band: dCas9 fragment. Bottom left. Upper band: pX334 Backbone fragment. |
 |
| Top right. Lowest band (blurred): CAG promoter fragment. |
Gel Extraction of digest fragments
| name |
ng/µl |
| Cas9-KRAB |
26.7 |
| dCas9 |
5.0 |
| pX334 Backbone |
14.4 |
| CAG promoter |
5.0 |
Ligation and transformation of intact pIG2004 and pIG2005
Ligation of pIG2005
| µl |
type |
| 11.2 |
CAG fragment |
| 4.4 |
dCas9 fragment |
| 1.4 |
pX334 backbone |
| 2 |
T4 ligase buffer |
| 1 |
T4 ligase |
Ligation of pIG2004
| µl |
type |
| 0.8 |
Cas9-KRAB fragment |
| 14.8 |
CAG fragment |
| 1.4 |
pX334 backbone |
| 2 |
T4 ligase buffer |
| 1 |
T4 ligase |
08. June
Evaluation of Transformations of pIG2005 and pIG2004
- pIG2004 (30 µl strikeout): 24 colonies
- pIG2005 (30 µl strikeout): 15 colonies
- 12 colonies of both approaches were striked out for further sequencing
09. June
Minipreps of pIG2004 and pIG2005
| name |
ng/µl |
| pIG2004 - No. 1 |
198.4 |
| pIG2004 - No. 2 |
203.6 |
| pIG2004 - No. 3 |
202.0 |
| pIG2004 - No. 4 |
187.3 |
| pIG2004 - No. 5 |
195.3 |
| pIG2004 - No. 6 |
211.2 |
| pIG2004 - No. 7 |
187.4 |
| pIG2004 - No. 8 |
201.1 |
| pIG2004 - No. 9 |
201.7 |
| pIG2004 - No. 10 |
206.3 |
| pIG2004 - No. 11 |
212.3 |
| pIG2004 - No. 12 |
174.6 |
| pIG2005 - No. 1 |
202.0 |
pIG2005 - No. 2 |
195.3 |
pIG2005 - No. 3 |
189.9 |
pIG2005 - No. 4 |
221.4 |
pIG2005 - No. 5 |
205.4 |
pIG2005 - No. 6 |
194.4 |
pIG2005 - No. 7 |
206.7 |
pIG2005 - No. 8 |
206.3 |
pIG2005 - No. 9 |
220.4 |
pIG2005 - No. 10 |
192.9 |
pIG2005 - No. 11 |
188.8 |
pIG2005 - No. 12 |
192.1 |
- Colonies No. 2 and 3 of pIG2004 were sent to sequencing, intending to validate (i) KRAB-insertion (ii) H840A conversion and (iii) an intact CAG promoter
- Colonies No. 1 and 4 of pIG2005 were sent to sequencing, intending to validate (i) H840A conversion and (ii) an intact CAG promoter
11. June
Evaluation of Sequencing results of pIG2004 and pIG2005
- pIG2004 clone No. 2 misses the KRAB fragment, but contains the CAG promoter
- pIG2004 clone No. 3 contains KRAB fragment, H840A and the CAG promoter - and will therefore be used for further studies
- pIG2005 clones No. 1 and 4 contain both H840 mutation and the CAG promoter
Restriction digest of pIG2004 and pIG2005 with BbsI
| µl |
type |
| 6 |
DNA |
| 4 |
NEB-Buffer 2.1 |
| 2 |
BbsI |
| Add to 40µl |
H2O |
 |
| F.l.t.r.: marker, pIG2004 undigested, pIG2004 digested, pIG2005 undigested, pIG2005 digested (2x) |
Gel Extraction of digested vectors pIG2004 and pIG2005
| name |
ng/µl |
| pIG2004 |
16.1 |
| pIG2005 |
18.8 |
12. June
Oligo annealing of target crRNAs
| µl |
type |
| 3 |
Oligo No. 1 |
| 3 |
Oligo No. 2 |
| 69 |
NEB-Buffer 2 |
- Samples were heated up to 95 degrees for 5 minutes. Gradient cool down to room temperature for 2 hours.
- Pair 1: oIG2010/oIG2011 - EMX1
- Pair 2: oIG2022/oIG2023 - Target 200 bp upstream of SEAP transcription initiation on pKM006
- Pair 3: oIG6008/oIG6009 - CMV
13-20. June
Ligation and transformation of crRNAs with Cas9-KRAB and dCas9
Ligation of pIG2013
| µl |
type |
| 15 |
Annealed EMX1 |
| 3 |
oIG2004 |
| 2 |
T4 ligase buffer |
| 1 |
T4 ligase |
Ligation of pIG2017
| µl |
type |
| 15 |
Annealed EMX1 |
| 3 |
oIG2005 |
| 2 |
T4 ligase buffer |
| 1 |
T4 ligase |
Ligation of pIG2019
| µl |
type |
| 15 |
Annealed SEAP target |
| 3 |
oIG2004 |
| 2 |
T4 ligase buffer |
| 1 |
T4 ligase |
Ligation of pIG2020
| µl |
type |
| 15 |
Annealed SEAP target |
| 3 |
oIG2005 |
| 2 |
T4 ligase buffer |
| 1 |
T4 ligase |
Ligation of pIG2011
| µl |
type |
| 15 |
Annealed CMV target |
| 3 |
oIG2004 |
| 2 |
T4 ligase buffer |
| 1 |
T4 ligase |
Ligation of pIG2015
| µl |
type |
| 15 |
Annealed CMV target |
| 3 |
oIG2005 |
| 2 |
T4 ligase buffer |
| 1 |
T4 ligase |
Evaluation of transformation results
- Except for pIG2020, colonies were grown in each case
- 6 colonies of pIG2011, pIG2013, pIG2015, pIG2017 and pIG2019 were respectively striked out
- All attempts had to be repeated at least twice, as BbsI digestion often lead to unspecific digestion of direct-repeats next to crRNA insertion spacer.
18-25. June
MIDI-preps of pIG2004, pIG2005, pIG2011, pIG2013, pIG2015, pIG2017, pIG2019 and also pIG9000
| name |
ng/µl |
| pIG2004 (Cas9-KRAB) |
400 |
| pIG2005 (dCas9) |
203 |
| pIG2011 (Cas9-KRAB vs. CMV) |
610 |
| pIG2013 (Cas9-KRAB vs. EMX1) |
609 |
| pIG2015 (dCas9 vs. CMV) |
460 |
| pIG2017 (dCas9 vs. EMX1) |
520 |
| pIG2017 (dCas9 vs. EMX1) |
520 |
| pIG2019 (Cas9-KRAB vs. SEAP) |
430 |
| pIG9000 (Cas9 vs. EMX1) |
460 |
- MIDIs had to be repeated three times
- During the first attempt, an unkown white precipitate was obtained - which could not be separated from the desired DNA.
- Within the second attempt, DNA was not adequately treated during isopropanol treatment (has to be shaken).
25.-27. June
Test of Cas9-KRAB on Western Blots
- Cas9-KRAB and dCas9, both targetting the CMV promoter were respectively co-transfected to HEK 293 cells with another plasmid, encoding CMV driven CNK.
- Both attempts were performed repeatedly, and cell lyses were than to be performed 12, 18, 36 and 42 hours post-transfection.
- Transfections were performed with PEI
- Lyses were performed with modified RIPA buffer
- Biological double attempts were respectively done
Transfection schemes for one 6-well plate
Cas9-KRAB vs. CMV of CNK (2x)
dCas9 vs. CMV of CNK (2x)
CNK negative control (2x)
Cas9-KRAB and dCas9 expression controls on one well
| name |
µg |
| pIG2004 |
1 |
| pIG2005 |
1 |
Results
 |
| ??? |
 |
| ??? |
- We expected a stronger decrease in CNK levels, when combining it with Cas9-KRAB than with dCas9
- Cas9-KRAB was able to highly efficiently repress CMV driven CNK expression
- Cas9-KRAB was expressed.
29. June
Test of Cas9-KRAB vs. CMV-driven GFP
- Transfection of pIG6000 (CMV::GFP) in combination with either pIG2011 (Cas9-KRAB + crRNA agaisnt CMV) or Mock DNA
- Cells were seeded previously seeded on 6-well plates
- Incubation for about 36 hours
dCas9 vs. CMV of GFP
| name |
µg |
| CMV-GFP |
1 |
| pIG2011 (Cas9-KRAB) |
2 |
| PEI |
9 |
 |
| Transfection scheme |
01. July
Fluorescence Microscopy Evaluation
 |
| Fluorescence Images |
ImageJ: Cells treated with GFP and with pRSET (Mock-DNA)
| Cell |
Size |
Intensity |
| 1 |
208 |
2920.9 |
| 2 |
146 |
3945.0 |
| 3 |
238 |
3064.8 |
| 4 |
166 |
3699.3 |
| 5 |
238 |
2071.3 |
| 6 |
192 |
2219.1 |
| 7 |
172 |
3819.9 |
| 8 |
228 |
3235.1 |
| 9 |
247 |
3510.0 |
| 10 |
146 |
3213.6 |
| 11 |
228 |
1977.4 |
| 12 |
224 |
3436.5 |
| 13 |
224 |
3436.5 |
| 14 |
136 |
1872.9 |
| 15 |
140 |
1611.4 |
ImageJ: Cells treated with GFP and Cas9-KRAB-DNA
| Cell |
Size |
Intensity |
| 1 |
268 |
2407.6 |
| 2 |
176 |
3329.2 |
| 3 |
217 |
1336.1 |
| 4 |
166 |
1138.5 |
| 5 |
102 |
2844.8 |
| 6 |
217 |
936.6 |
| 7 |
148 |
1236.4 |
| 8 |
214 |
1032.9 |
| 9 |
177 |
1566.6 |
| 10 |
96 |
1461.9 |
| 11 |
228 |
1977.4 |
| 12 |
938 |
3371.7 |
| 13 |
102 |
992.9 |
| 14 |
210 |
982.1 |
| 15 |
112 |
1016.6 |
- Mean of GFP fluorescence intensity of cells treated with Cas9-KRAB is 1409.4 (SD: 880.6), cells co-transfected with Mock-DNA revealed 2718 (SD: 786.6)
- Results should be of low validity, but are potentially indicative towards a repressive effect of Cas9-KRAB
02. July
MIDI-prep of pIG2005, pIG2019 and pRSET (Mock-DNA)
Failed in each case, for unknown reasons (white salt fall-out after elution from column). Attempt will be repeated on the next day.
03. July
MIDI-prep of pIG2005, pIG2019 and pRSET (Mock-DNA)
| name |
ng/µl |
| pIG2005 (dCas9) |
470 |
| pIG2019 (Cas9-KRAB vs. SEAP target) |
430 |
| pRSET (Mock DNA) |
790 |
04. July
Transfection of pIG2019 in combination with pKM006 (SEAP plasmid) and pSAM200 (SV40::tetR-VP16)
- Biological tetraplicates (each attempt was repeated independently, for four different wells of one 6-well plate
- Negative control: pKM006 (500 ng) + Mock-DNA (2500 ng)
- Positive control: pKM006 (500 ng) + pSAM200 (500 ng) + Mock-DNA (2000 ng)
- Cas9-KRAB test: pKM006 (500 ng) + pSAM200 (500 ng) + pIG2019 (2000 ng)
07. July
SEAP measurement
- 300µl of medium supernatants were removed from each well
- Samples were heated to 65°C for 30 minutes
- Centrifugation at 600 rpm (table top) for 1 minute
- 80µl of each supernatant was transfered to one well of a 96-well plate, technical triplicates for each sample were pipetted
- 100µl 2x SEAP Buffer was added to each well
- 20µl pNPP substrate was added to each well
- Air bubbles were quickly removed
- Measurement was taken with a plate reader, for two consecutive hours with an interval lenght of one minute. Detection wavelength was 405 nm
 |
| SEAP Results |
01. September
Test of Cas9-KRAB vs. CMV-driven GFP, Repetition of the Experiment from 29. June on 24 well plates
dCas9 vs. CMV of GFP
| name |
µg |
| CMV-GFP |
0,04 |
| pIG2011 (Cas9-KRAB) |
0,46 |
| PEI |
1,5 |
dCas9 vs. CMV of GFP
| name |
µg |
| CMV-GFP |
0,04 |
| pRSET (Mock-DNA) |
0,46 |
| PEI |
1,5 |
03. September
Fluorescence Microscopy of KRAB-mediated GFP-repression
 |
| Results of GFP repression. Experiment will be repeated with off-target controls and parallel Flow Cytometry analysis. |
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