Team:Freiburg/Notebook/lab repression

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Effector - Repression

21. April

Minipreps of pKM006, pKM102 & pSAM200

name	ng/µl
pSAM200	320.1
pKM006	113.3
pKM102	180.0

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)

name	µg
CNK	6
pIG2011	12

dCas9 vs. CMV of CNK (2x)

name	µg
CNK	6
pIG2015	12

CNK negative control (2x)

name	µg
CNK	6

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