SpEDIT CRISPR/Cas9 method Full protocol

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CRISPR/Cas9 protocol (based on Rodríguez-López et al 2017 v3 with Plasmid construction from Torres-Garcia et al. 2020) For old protocol see this page.

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 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. (dilute it 100x times: 5µl of the guide RNA + 500µl H2O)

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!
  • If you have some (1-50%) GFP colonies, mark them.
  • Pick 4 colonies from the LB plate and grow them in 5ml LB+amp (100µg/ml final concentration)
  • Incubate overnight at 37°C in the shaker.

Plasmid purification and -80°C glycerol stock

  • Take 0.5 ml culture and add 0.5 ml sterile 50% Glycerol (in fridge), mix and store at -80°C.
    • Print a label with the (temporary) plasmid and stick on the tube
    • Put the tube in the appropriate box
    • Add the plasmid and its location to the -80°C list
  • With the remaining 4ml cells do a Macherey-Nagel miniprep according to the manufacturer’s protocol with modifications as indicated (use 2x 2ml cells etc.).
  • Do a sequencing PCR reaction with the plasmid
  • Freeze the rest of the plastmid at -20°C

Verify insert into 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 yeast cell 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 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 -20°C 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.

  1. Thaw by heating (use the 42°C bath) and then put on ice:
    • ssDNA solution (salmon sperm DNA, 10μg/μl ; in freezer 7)
    • 50% PEG4000 solution
    • Competent fission yeast cells
  2. Thaw on ice:
    • HR template repair DNA
    • sgRNA plasmid (concentration should be ~200ng/μl)
  3. 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
  4. Add 145μl PEG4000 and mix by pipetting up and down a few time
  5. Immediately incubate for 15min @ 42°C.
  6. Centrifuge the cells at 1600 x g for 3 min at room temperature (RT).
  7. Remove supernatant and re-suspend the cells in 1 ml
    • EMM-N or
    • EMM when working with h90 or an other homothallic strain
    Reduce the added supplements (e.g. leucine, adenine etc) to 10% of the standard amount.
  8. Incubate at RT for ~16h
  9. Spin down the cells at 1600 x g for 3 min, RT and remove supernatant
  10. Resuspend cells in 100 μl water and spread them on YES plates with 100 μg/μl Nourseothricin.
  11. 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 MasterMix for 8 MasterMix 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.