Difference between revisions of "SpEDIT CRISPR/Cas9 method Full protocol"
Line 11: | Line 11: | ||
* Copy the Deletion Checking primers (again same for either sgRNA) | * Copy the Deletion Checking primers (again same for either sgRNA) | ||
One set should thus contain 8 primers (2x HR, 2x Check, 4x sgRNA). | One set should thus contain 8 primers (2x HR, 2x Check, 4x sgRNA). | ||
− | Ask Cristina or Bart to order these from metaBion. Order all desalted at the lowest amount that is allowed for the length of the oligo. | + | Ask Cristina or Bart to order these from metaBion. Order all desalted at the lowest amount that is allowed for the length of the oligo, liquid 100µM dissolved. |
== Create plasmids for transformation == | == Create plasmids for transformation == | ||
− | A ''Bsa''I | + | A ''Bsa''I [Golden Gate] cut-ligation is performed to introduce the right sgRNA template into a backbone plasmid. There are in total 3 different plasmids (gifts from the Allshire lab) that differ in the yeast resistance marker in the backbone: |
* pBN305 spedit kanMX CRISPR/Cas9 GFP GG - pLSB-kanMX [amp] --> G418 | * pBN305 spedit kanMX CRISPR/Cas9 GFP GG - pLSB-kanMX [amp] --> G418 | ||
* pBN306 spedit natMX CRISPR/Cas9 GFP GG - pLSB-natMX [amp] --> nourseothricin | * pBN306 spedit natMX CRISPR/Cas9 GFP GG - pLSB-natMX [amp] --> nourseothricin | ||
Line 20: | Line 20: | ||
===Setup GG ligation=== | ===Setup GG ligation=== | ||
+ | Make a dilution of the 100µM stocks for the two primers for the sgRNA guides: | ||
+ | * 10 µl Fwd | ||
+ | * 10 µl Rev | ||
+ | * 80 µl H2O | ||
+ | |||
+ | Take 10µl of the mix in a PCR tube and place in PCR thermocycler | ||
+ | Run touchdown program: | ||
{| class="wikitable" | {| class="wikitable" | ||
− | ! | + | !Number of cycles |
− | ! | + | !Temperature |
+ | !Duration | ||
|- | |- | ||
− | | | + | |1X || 95°C || 10 s || |
|- | |- | ||
− | | | + | |40X || 95°C || 5 s || reduce temp after 1 cycle by 2°C every cycle |
|- | |- | ||
− | | | + | | || 7°C || Inf |
+ | |} | ||
+ | |||
+ | Dilute your now double stranded DNA fragment that is the template for the Guide RNA. | ||
+ | |||
+ | {| class="wikitable" | ||
+ | |Diluted guide RNA fragment||1.0||μl | ||
+ | |- | ||
+ | |Backbone at 20fmol/µl||1.0||μl | ||
+ | |- | ||
+ | |BsaI-HF V2 (5 units)||0.75||μl | ||
|- | |- | ||
− | | | + | |T4 ligase (NEB)||0.75||μl |
|- | |- | ||
− | | | + | |CutSmart 10X||1.5||µl |
|- | |- | ||
− | | | + | |ligase buffer (avoid freeze-thaw cycles to protect the ATP)||1.5||μl |
− | |- | + | |- |
− | | | + | |PCR grade H2O||8.5||µl |
|} | |} | ||
− | + | Incubate in a thermocycler using 'GoldenGate' program: | |
− | |||
{| class="wikitable" | {| class="wikitable" | ||
!Number of cycles | !Number of cycles | ||
Line 46: | Line 63: | ||
!Duration | !Duration | ||
|- | |- | ||
− | | | + | |1X || 37°C || 10 min |
|- | |- | ||
− | | | + | |40X || 37°C || 3 min |
|- | |- | ||
− | | || | + | | || 16°C || 3 min |
|- | |- | ||
− | | || | + | |1X || 37°C || 5 min |
|- | |- | ||
− | | | + | | || 50°C || 5 min |
+ | |- | ||
+ | | || 80°C || 5 min | ||
|- | |- | ||
| || 7°C || Inf | | || 7°C || Inf | ||
Line 62: | Line 81: | ||
We use OneShot TOP10 Chemically competent ''E. coli'' cells (comparable to DH5α) that are to be found in the -80°C freezer (Parsch freezer). <BR> | We use OneShot TOP10 Chemically competent ''E. coli'' cells (comparable to DH5α) that are to be found in the -80°C freezer (Parsch freezer). <BR> | ||
− | |||
− | Do transformation using the long version of the | + | Do transformation using the long version of the manufacturer's protocol: |
− | *Thaw the cells on ice | + | *Thaw the cells on ice. |
− | *Add DNA to the cells and place back on ice for 30 min: | + | *Add 7.5µl plasmid ligate DNA to the cells and place back on ice for 30 min: |
− | |||
*Heatshock the cells for 30sec at 42°C | *Heatshock the cells for 30sec at 42°C | ||
Line 74: | Line 91: | ||
*Preheat [[LB]] plates with 75 μg/ml [[Ampicillin]] (1:1000 dilution of 100mg/ml stock) to 37°C. | *Preheat [[LB]] plates with 75 μg/ml [[Ampicillin]] (1:1000 dilution of 100mg/ml stock) to 37°C. | ||
*Spread the 100 μl cells on the preheated LB+amp plates and incubate overnight at 37°C. | *Spread the 100 μl cells on the preheated LB+amp plates and incubate overnight at 37°C. | ||
− | *:the rest of the cells can be left in the fridge to plate out if needed. | + | *:the rest of the cells can be left in the fridge (=5°C) to plate out the next day if needed. |
*After one day the plates should show colonies. | *After one day the plates should show colonies. | ||
=== Pick and check the transformed plasmid=== | === Pick and check the transformed plasmid=== | ||
− | + | During the GG reaction the GFP marker in the backbone should have been replaced by the short 20bp long fragment that is the template for our sgRNA. Colonies that shine GFP are thus not correct. | |
+ | * Check under fluorescence stereo microscope (2nd floor) for GFP fluorescence of the colonies. | ||
+ | ** if all are green, something went wrong. Check your design (are the overhangs correct?) and the dilution (too high insert concentration can affect GG) | ||
+ | ** if none are green, something might have gone wrong. We expect the GG to be efficient, but generally not 100% efficient! | ||
+ | |||
* Pick some colonies from the LB plate and grow each in a well of sterile PCR strips or a 96 well plate with 50 μl LB+Amp (75 μg/ml). | * Pick some colonies from the LB plate and grow each in a well of sterile PCR strips or a 96 well plate with 50 μl LB+Amp (75 μg/ml). | ||
* Incubate overnight at 37°C. | * Incubate overnight at 37°C. |
Revision as of 16:08, 9 May 2022
CRISPR/Cas9 protocol (based on [Rodríguez-López et al 2017 v3] with Plasmid construction from [Torres-Garcia et al. 2020]) For old protocol see [CRISPR_Cas9_Full_protocol here].
Design and order primers
- Go to the Bähler-lab website and use the crispr4p methods from the resource section to design primers.
- Selection the region or gene and run the script
- Choose 2 of the suggested sgRNAs and introduce them in the template sequence on [[https://www.benchling.com/ Benchling (search for 'GG_sgRNA_template spedit')
- generate two primers of this full fragment: one forward and one reverse.
- Copy the Homologous Recombination template primers (these are the same for either sgRNA) if you are performing a deletion.
- Copy the Deletion Checking primers (again same for either sgRNA)
One set should thus contain 8 primers (2x HR, 2x Check, 4x sgRNA). Ask Cristina or Bart to order these from metaBion. Order all desalted at the lowest amount that is allowed for the length of the oligo, liquid 100µM dissolved.
Create plasmids for transformation
A BsaI [Golden Gate] cut-ligation is performed to introduce the right sgRNA template into a backbone plasmid. There are in total 3 different plasmids (gifts from the Allshire lab) that differ in the yeast resistance marker in the backbone:
- pBN305 spedit kanMX CRISPR/Cas9 GFP GG - pLSB-kanMX [amp] --> G418
- pBN306 spedit natMX CRISPR/Cas9 GFP GG - pLSB-natMX [amp] --> nourseothricin
- pBN307 spedit hphMX CRISPR/Cas9 GFP GG - pLSB-hphMX [amp] --> hygromycin
Setup GG ligation
Make a dilution of the 100µM stocks for the two primers for the sgRNA guides:
- 10 µl Fwd
- 10 µl Rev
- 80 µl H2O
Take 10µl of the mix in a PCR tube and place in PCR thermocycler Run touchdown program:
Number of cycles | Temperature | Duration | |
---|---|---|---|
1X | 95°C | 10 s | |
40X | 95°C | 5 s | reduce temp after 1 cycle by 2°C every cycle |
7°C | Inf |
Dilute your now double stranded DNA fragment that is the template for the Guide RNA.
Diluted guide RNA fragment | 1.0 | μl |
Backbone at 20fmol/µl | 1.0 | μl |
BsaI-HF V2 (5 units) | 0.75 | μl |
T4 ligase (NEB) | 0.75 | μl |
CutSmart 10X | 1.5 | µl |
ligase buffer (avoid freeze-thaw cycles to protect the ATP) | 1.5 | μl |
PCR grade H2O | 8.5 | µl |
Incubate in a thermocycler using 'GoldenGate' program:
Number of cycles | Temperature | Duration |
---|---|---|
1X | 37°C | 10 min |
40X | 37°C | 3 min |
16°C | 3 min | |
1X | 37°C | 5 min |
50°C | 5 min | |
80°C | 5 min | |
7°C | Inf |
Transformation of competent E. coli cells for sgRNA
We use OneShot TOP10 Chemically competent E. coli cells (comparable to DH5α) that are to be found in the -80°C freezer (Parsch freezer).
Do transformation using the long version of the manufacturer's protocol:
- Thaw the cells on ice.
- Add 7.5µl plasmid ligate DNA to the cells and place back on ice for 30 min:
- Heatshock the cells for 30sec at 42°C
- Add 250μl 37°C SOC or LB
- Incubate these tubes with shaking at 37°C for 1 hour.
- Preheat LB plates with 75 μg/ml Ampicillin (1:1000 dilution of 100mg/ml stock) to 37°C.
- Spread the 100 μl cells on the preheated LB+amp plates and incubate overnight at 37°C.
- the rest of the cells can be left in the fridge (=5°C) to plate out the next day if needed.
- After one day the plates should show colonies.
Pick and check the transformed plasmid
During the GG reaction the GFP marker in the backbone should have been replaced by the short 20bp long fragment that is the template for our sgRNA. Colonies that shine GFP are thus not correct.
- Check under fluorescence stereo microscope (2nd floor) for GFP fluorescence of the colonies.
- if all are green, something went wrong. Check your design (are the overhangs correct?) and the dilution (too high insert concentration can affect GG)
- if none are green, something might have gone wrong. We expect the GG to be efficient, but generally not 100% efficient!
- Pick some colonies from the LB plate and grow each in a well of sterile PCR strips or a 96 well plate with 50 μl LB+Amp (75 μg/ml).
- Incubate overnight at 37°C.
- Transfer 25 μl of each well to a fresh plate with 50 μl LB+Amp and place at 37°C.
- Boid the remaining 25 μl in a PCR machine at 98°C for 10min.
- Leave on ice for a bit.
- Add 3μl Loading Dye and load a much of the mix on a gel with Ethidium Bromide. If you use another dye such as RotiSafe or GelRed, you will not get a result! If you don't want to use EtBr, you can always use this longer protocol.
- Choose the colonies that show fully intact plasmids and continue with them in the next step.
Plasmid purification and -80°C glycerol stock
- Grow two cultures of bacteria with the correct plasmid clone in 5ml LB + 75 μg/ml Ampicillin at 37°C overnight.
- Take 0.5 ml culture and add 0.5 ml sterile 50% Glycerol (in fridge), mix and store at -80°C.
- Add the plasmid to the list in the binder on Bart’s bench
- Label the tube well with the number from the list
- Put the tube in the appropriate box and add the location to the too
- With the remaining cells do a Zymo miniprep according to the manufacturer’s protocol.
- Do a sequencing PCR reaction with the plasmid
- With the remaining plasmids:
- Make a few aliquots and store these at -20°C. The plasmid when not in the E. coli is very unstable and should be thawed and frozen as little as possible.
Test plasmid by sequencing
In this reaction we sequence the cloned sgRNA region on the plasmid. After the reaction the sample can be ran on an ABI sequencer to obtain the DNA sequence.
Do sequencing reaction: | |
---|---|
1 μl | Miniprep |
1 μl | Primer M13F (10 μM) |
5 μl | H2O |
2 μl | BigDye sequencing mix (-20°C) |
1 μl | Sequencing buffer 5X (fridge, and yes, we use 5X to get a final concentration of 0.5X) |
Run using “bigdye” program (in all machines under e.g. Gaby, Hilde or Simone)
Number of cycles | Temperature | Duration |
---|---|---|
1 X | 96°C | 1 min |
40X | 96°C | 10 sec |
50°C | 15 sec | |
60°C | 4 min | |
1X | 7°C | Inf |
Homologous recombination DNA
This is a fragment of DNA that will be co-transformed into the yeastcell and that will be used as a template to repair the DNA after the Cas9-induced double strand break. For knockout of a gene or deletion of a genomic sequence, use the HR primers as designed in step 1. These two primers (HRFw and HRRv) will be used in a PCR reaction to generate an 180bp long fragment. Primers should be diluted 1:10 in water (e.g. 10 μl + 10 μl + 80 μl for HRFw, HRRv and H2O, respectively). Always work on ice.
Do sequencing reaction: | Total volume 50 μl |
---|---|
Phusion 5X HF buffer | 10 μl |
H2O | 36.5 μl |
Primer mix (10 μM/ primer) | 2 μl |
dNTPs (10 μM each) | 1 μl |
Phusion polymerase | 0.5 μl |
Run using program (bart -> pombe -> HR PCR)
Number of cycles | Temperature | Duration |
---|---|---|
1 X | 98°C | 2 min |
25X | 98°C | 10 sec |
55°C | 10 sec | |
72°C | 30 sec | |
1X | 72°C | 5 min |
7°C | Inf |
Store fragment at -20oC until needed.
Transformation of chemically competent yeast cells
Chemically competent yeast cells are available for some pombe strains in 50μl aliquots in the -80°C. If other strains are required, use the protocol Synchronized Competent Yeast Cells.
- Thaw by heating (use the 43°C bath) and then put on ice:
- Thaw on ice:
- HR template repair DNA
- sgRNA plasmid (concentration should be ~200ng/μl)
- Add to the competent cells in this order:
- 2 μl ssDNA (mix by carefully flicking the tube)
- 10 μl HR template
- 10 μl sgRNA plasmid
- Add 145μl PEG4000 and mix by pipetting up and down a few time
- Immediately incubate for 15min @ 43°C.
- Centrifuge the cells at 1600 x g for 3 min at room temperature (RT).
- Remove supernatant and re-suspend the cells in 1 ml
- Reduce the added supplements (e.g. leucine, adenine etc) to 10% of the standard amount.
- Incubate at RT for ~16h
- Spin down the cells at 1600 x g for 3 min, RT and remove supernatant
- Resuspend cells in 100 μl water and spread them on YES plates with 100 μg/μl Nourseothricin.
- Incubate at 32°C for at least 4 days (or even longer!)
Select resistant yeast cells
After incubation, large, small, and tiny colonies should become visible on the plate. We are interested in the smallest colonies.
The plasmid is very costly —most likely due to expression of Cas9— for the yeast cells which stunts their growth. The smallest colonies are resistant and express Cas9, which is what we’re interested in. The larger colonies are still resistant, but probably lost part of the plasmid, incl Cas9, so that’s no good.
- Pick 16 colonies, and streak each out on YES to obtain single colonies, this allows cells to lose the costly sgRNA plasmid.
- Pick a colony and patch the colony on a fresh YES plate.
Verify deletion or replacement by PCR
Perform a PCR on the colonies selected at Select resistant yeast cells.
Prepare mastermix, divide over PCR reaction tubes/wells and add the yeast cells to the mix using pipette tips (so not toothpicks). For the reaction we use the deletion Check primers designed at step 2. The result should show a shorter band if a deletion was successful. Also run a positive control of untransformed cells.
Do sequencing reaction: | per samples | MMix for 8 | MMix for 16 |
---|---|---|---|
TopTaq PCR Buffer 10X | 2.5 μl | 21 μl | 42 μl |
MgCl2 (25mM) | 0.5 μl | 4.2 μl | 8.4 μl |
H2O | 12.25 μl | 102.9 μl | 205.8 μl |
Primer mix (10 μM/ primer) | 1 μl | 8.4 μl | 16.8 μl |
Q solution 5X | 5 μl | 42 μl | 84 μl |
CoralLoad 10X | 2.5 μl | 21 μl | 41 μl |
dNTPs (10 μM each) | 1 μl | 8.4 μl | 16.8 μl |
TopTaq polymerase (5U/μl) | 0.2 μl | 1.7 μl | 3.4 μl |
Run using program (bart -> pombe -> colony PCR)
Number of cycles | Temperature | Duration |
---|---|---|
1 X | 94°C | 2 min |
35X | 94°C | 30 sec |
52°C | 30 sec | |
72°C | 2 min 30 sec | |
1X | 72°C | 7 min |
7°C | Inf |
- Run the product on a 1% TAE gel at 10 V/cm for 20 min.
- Continue with the strain(s) with the appropriate PCR product.
- Grow the cells from the colony in 3ml liquid YES overnight
- Make a -80°C glycerol stock for future reference.
- Add the strain(s) to the “pombe strain list” in the binder on Bart’s desk and add all the required information.
- Label the tube well with the number from the list
- Put the tube in Box 4 of Rack Pombe 1 and add the -80°C location to the list too.