SpEDIT CRISPR/Cas9 method Full protocol

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.  

Create plasmids for transformation

A BsaI GoldenGate 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

Do sequencing reaction	Total volume 25 μl
Plasmid (aliquot)	1 μl
Phusion 5X HF buffer	5 μl
H2O	16.8 μl
Primer mix (10 μM of sgRNA-Fw and sgRNA-Rv primers each)	1 μl or 1μl of each if no mix
dNTPs (10 μM each)	0.5 μl
DMSO 100%	0.5 μl
Phusion polymerase	0.25 μl

Run using program (bart -> cloning -> sgRNA)

Number of cycles	Temperature	Duration
1 X	98°C	2 min
25X	98°C	10 sec
	60°C	30 sec
	72°C	5 min 30 sec
1X	72°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).
For each transformation, we use 25μl. Each tube in the -80 can thus be used for 2 transformations.

Do transformation using the long version of the manufacturer’s protocol:

• Thaw the cells on ice (and split over 2 tubes to get 25μl cells, do not pipette more than once as cells are very sensitive).
• Add DNA to the cells and place back on ice for 30 min:
  • 5 μl PCR product of an sgRNA cloned linear plasmid

• 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 to plate out if needed.

• After one day the plates should show colonies.

Pick and check the transformed plasmid

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Figure from Rodríguez-López et al 2017. https://wellcomeopenresearch.org/articles/1-19/v3 The red arrows in the gel indicate samples that are not correct.

• 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.

1. Thaw by heating (use the 43°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 @ 43°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	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.