Jul 23, 2025

Public workspaceCRISPR-Cas9 genome editing in Corallochytrium limacisporum

DOI
https://dx.doi.org/10.17504/protocols.io.kxygxq6q4v8j/v1
  • Patricia S. Ara1,
  • Elena Casacuberta1,
  • Claudio Scazzocchio2,3,
  • Iñaki uiz-Trillo1,4,
  • Sebastián Najle1,5
  • 1Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Barcelona, Spain;
  • 2Department of Life Sciences, Imperial College London, London, UK;
  • 3Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France;
  • 4ICREA, Pg. Lluís Companys 23, 08010 Barcelona, Spain;
  • 5Centre for Genomic Regulation (CRG), Barcelona Institute for Science and Technology, Barcelona, Spain.
Patricia S Ara
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygxq6q4v8j/v1
Protocol Citation: Patricia S. Ara, Elena Casacuberta, Claudio Scazzocchio, Iñaki uiz-Trillo, Sebastián Najle 2025. CRISPR-Cas9 genome editing in Corallochytrium limacisporum. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxq6q4v8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 10, 2025
Last Modified: July 23, 2025
Protocol Integer ID: 219832
Keywords: Genome editing, Corallochytrium limacisporum, Unicellular holozoans, CRISPR-Cas9, cas9 genome editing in corallochytrium limacisporum, genome editing in corallochytrium limacisporum, cas9 genome editing, genome editing, cas9 rnp complex, crispr, corallochytrium limacisporum, unicellular marine eukaryote, resistance to rapamycin,
Funders Acknowledgements:
MICIU/ AEI /10.13039/501100011033
Grant ID: PID2020-120609GB-I00
MICIU/AEI /10.13039/501100011033
Grant ID: PID2023-153273NB-I00
Moore Foundation
Grant ID: and MMI Experimental Model Systems grant #4973.01
MICIU/AEI/10.13039/501100011033
Grant ID: PRE2018-085438
Abstract
This protocol walks you through how to achieve genome editing in Corallochytrium limacisporum, a unicellular marine eukaryote which is closely related to animals. Here we describe how to achieve resistance to rapamycin or carboxin. In brief, the cells are Nucleofected, to introduce a sgRNA-Cas9 RNP complex. Next, the cells are selected on agar plates and isolated for genotyping.

Materials
  • Marine Broth (BD Difco #2216)
  • Marine Agar (Scharlau #01-291)
  • T-25 flasks (Corning #430639)
  • 90 mm cell culture dishes (Falcon #353002)
  • 12-well plate (Corning #3513)
  • 96-well plate (Nunc #167008)
  • Cell scraper (Corning #3010)
  • 5 mm glass beads (Sigma-Aldrich #18406)
  • P3 Primary Cell 4D-Nucleofector X Kit (Lonza #V4XP-3032)
  • Amaxa ® 4D-Nucleofector ®
  • EnGen Spy Cas9 NLS (New England Biolabs, #M0646M)
  • PBS (Sigma #P5368-10PAK)
  • Rapamycin Sigma-Aldrich #37094
  • Carboxin Sigma-Aldrich #45371
  • Ca-Mg Free Sea Water: NaCl 460 mM, NaHCO3 2.15 mM, KCl 10.7 mM, Na2SO4 7 mM, Tris-HCl pH 8.0 10 mM
Troubleshooting
Before start
  • C. limacisporum is prone to contamination as its growth medium is very rich, always proceed under sterile conditions (laminar flow cabinet) to avoid contamination.
  • Unless otherwise stated, C. limacisporum is grown in Marine Broth, its usual growth medium.
  • All reagents and their references can be found in the Materials section.
Seed cells for transfection
20m
Prepare a T-25 flask of C. limacisporum so that it is growing exponentially on the day of transfection. Usually, Amount100-200 µL from a confluent culture in a 25 cm2 flask withAmount5 mL of Marine Broth works well.

20m
Overnight
Incubate the cells at Temperature23 °C DurationOvernight in an incubator without agitation.

Transfection
40m
Before starting, prepare the following reagents and keep them on ice:
  • 1X Filter-sterilized PBS
  • Lonza nucleofector strip (part of P3 Primary Cell 4D-Nucleofector X Kit)
  • Lonza P3 Buffer (part of P3 Primary Cell 4D-Nucleofector X Kit)
  • Repair DNA template (if needed)
Note
Ensure that the supplement has been added to the Lonza P3 buffer.
Supplemented Lonza P3 Buffer expires 3 months after preparation. To avoid wasting P3 buffer, aliquot the components to prepare 1/3 of the volume:
  • Amount225 µL P3 solution
  • Amount50 µL supplement

Prepare RNP complex (sgRNA+Cas9) mix. One mix should be prepared spearately for each sgRNA to be used. When adding the final component, gently swirl it with the pipette tip and incubate at RT for at least 30 min.
RNP mix:
  • Amount10 µL Lonza P3 Buffer
  • Amount2.5 µL sgRNA at Concentration60 micromolar (µM)
  • Amount2 µL EnGen Spy Cas9 NLS
Proceed with the next steps during the Duration00:30:00 incubation.
Note
For co-transfection experiments, prepare the two mixes independently:
Mix 1:
  • 5 µL of P3 buffer
  • 2 µL EnGen Spy Cas9 NLS
  • 2.5 µL sgRNA1

Mix 2:
  • 5 µL of P3 buffer
  • 2 µL EnGen Spy Cas9 NLS
  • 2.5 µL sgRNA2


30m
Scrape and homogenize the cells, then determine the concentration by counting a 1:100 dilution of the homogenized cell suspension using a Neubauer chamber.
Pellet 1.5 x 106 cells per reaction at Centrifigation2000 x g, 00:05:00


Note
Cells for multiple transfections can be pelleted together in the same tube.


Note
During centrifugation: Prepare a 12-well plate with 1 mL of Marine Broth for every transfection

5m
Remove supernatant from pelleted cells and wash the pellet withAmount100 µL ice-cold sterile PBS.

Spin the cells again Centrifigation2000 x g, 00:05:00

Note
During centrifugation: Prepare the Lonza nucleofector.
Turn on Lonza nucleofector, select the vessel (strip) and select the wells that are going to be used.
- Solution: Primary cell P3
- Pulse Code: EN138
5m
Remove supernatant from the washed cells and resuspend in Amount6.5 µL ice-cold P3 buffer for every transfection that will be done.

Note
Don't leave the cells in P3 buffer for longer than necessary.

Prepare mix for nucleofection:
Amount14.5 µL RNP mix Go to
Amount6.5 µL cells resuspended in P3 Go to
Amount2 µL repair DNA template (if needed)


Note
For co-transfection with two guides:
  • 9.5 µL RNP mix 1
  • 9.5 µL RNP mix 2
  • 6.5 µL cells resuspended in P3
  • 2 µL repair template (if needed)

Note
When doing HDR, different length repair templates can be used at 100 uM. No repair template is necessary for NHEJ.

Add transfection mixes to the Lonza strip, place the strip in the machine and apply EN138 pulse
Quickly remove strip from Lonza and addAmount80 µL Marine Broth to allow the cells to recover

Transfer transfected cells to 12-well plate with 1 mL Marine Broth
Grow cells at Temperature23 °C DurationOvernight for selection on rapamycin, or Duration48:00:00 for selection on carboxin

Plating onto Marine Agar
Melt Amount20 mL of Marine Agar (Scharlau #01-291) for every plate that you are going to prepare.

Note
If you are using rapamycin always use fresh plates and wait until it is quite cool to the touch but not solidifying yet, as rapamycin is very sensitive to heat. Carboxin plates can be prepared in advance and stored at 4oC for up to a week.

Add selection drug to the agar and addAmount20 mL agar per dish. Allow them to solidify until it is dry.
Note
Rapamycin selection concentration: 125 ng/mL
Carboxin selection concentration: 30 µg/mL


Scrape the transfected wells and add Amount100-200 µL of the culture onto the agar.
Note
If pelleting a larger volume, spin the cells down first and resuspend the pellet in 100 µL of Marine Broth first.

Add 3-4 glass beads (Sigma-Aldrich #18406) and shake the plate until the agar is completely dry. Gently remove the glass beads by pouring them from the plate. Seal the plate with parafilm.
Incubate the plates at Temperature23 °C for 10-14 days.
Note
Monitor the plates to ensure contaminants don't appear, these will usually appear faster than C. limacisporum colonies. C. limacisporum will usually start to be detectable after around 5-7 days and appear as very small, transparent colonies. Eventually, they will become round and pink.

Colony picking and streaking
Prepare Marine Agar plates containing the selection drug as above Go to and label them with the approximate number of colonies that have appeared, or however many you want to isolate and screen.

Gently pick the colonies by touching them with a sterile pipette tip and gently streaking onto a fresh agar plate labelled with clone identifers. Seal the plate with parafilm.
Incubate the plates at Temperature23 °C allowing the streaks to grow for 2-3 days until the streak is visible.

Transfer clones to liquid medium
Once the streak is visible, pick a part of the streak using a sterile tip and place it in Amount100 µL Marine Broth with selecting drug in a 96-well plate.

Allow the cells to grow by incubating the plates at Temperature23 °C for 2-3 days before genotyping.

Genotyping
Pipette up and down to resuspend cells in the 96-well plate.
Transfer Amount50 µL of the culture to a tube. Fresh Marine Broth can be added to the well to maintain the culture if needed, otherwise they can also be retrieved from the streaked agar plates.
Note
50 µL of culture in a 0.2 mL tube or PCR strip is enough to genotype with, but larger volumes can be used by scaling up the amount of Ca-Mg free sea water used to wash the cells in step 28.


Pellet the cells atCentrifigation2500 x g, 00:03:00 . If using PCR strips, use a Minispin with strip adaptor for 3 minutes.

Note
From this step forward, sterile environment is no longer required.

Remove the supernatant and wash the cells with Amount100 µL Ca-Mg free sea water. Repeat the wash once.

Note
Ca-Mg free sea water:
NaCl 460 mM, NaHCO3 2.15 mM, KCl 10.7 mM, Na2SO4 7 mM, Tris-HCl pH 8.0 10 mM


After the second wash, heat the cell suspension for Duration00:10:00 atTemperature95 °C in a Thermoblock or PCR machine.

Spin down cell remains atCentrifigation2500 x g, 00:05:00 and use Amount1-2 µL of the supernatant as template in genotyping PCR.
Note
Lysed cells can be stored at 4ºC before genotyping for up to a week.