Dec 12, 2019

Public workspaceCRISPR/Cas9 based knockout generation in Aurantiochytrium limacinum (ATCC MYA-1381)

DOI
dx.doi.org/10.17504/protocols.io.baeyibfw
  • 1University of Illinois at Urbana-Champaign;
  • 2State University of New York at Stony Brook
  • Collier Lab
Anbarasu Karthikaichamy
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DOI: dx.doi.org/10.17504/protocols.io.baeyibfw
Protocol Citation: Anbarasu Karthikaichamy, Jackie Collier 2019. CRISPR/Cas9 based knockout generation in Aurantiochytrium limacinum (ATCC MYA-1381). protocols.io https://dx.doi.org/10.17504/protocols.io.baeyibfw
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: December 11, 2019
Last Modified: December 12, 2019
Protocol Integer ID: 30904
Keywords: Stramenopile, Thraustochytrid, Aurantiochytrium, CRISPR, Protists, Electroporation
Abstract
Transformation protocol for electroporation of CRISPR/Cas9 ribonucleoprotein (RNP) complex in urantiochytrium limacinum (ATCC MYA-1381; Stramenopile/ Heterokont, Thraustochytrid).
Materials
MATERIALS
ReagentNEPA21 Super ElectroporatorNEPAGENE
ReagentEnGen® sgRNA Synthesis Kit S. pyogenesNew England BiolabsCatalog #E3322S
ReagentCas9 Nuclease S. pyogenesNew England BiolabsCatalog #M0386S
ReagentMonarch® RNA Cleanup KitNew England BiolabsCatalog #T2050S
STEP MATERIALS
ReagentEnGen® sgRNA Synthesis Kit S. pyogenesNew England BiolabsCatalog #E3322S
ReagentCas9 Nuclease S. pyogenesNew England BiolabsCatalog #M0386S
ReagentMonarch® RNA Cleanup KitNew England BiolabsCatalog #T2050S
Protocol materials
ReagentEnGen® sgRNA Synthesis Kit S. pyogenesNew England BiolabsCatalog #E3322S
ReagentCas9 Nuclease S. pyogenesNew England BiolabsCatalog #M0386S
ReagentMonarch® RNA Cleanup KitNew England BiolabsCatalog #T2050S
ReagentNEPA21 Super ElectroporatorNEPAGENE
ReagentEnGen® sgRNA Synthesis Kit S. pyogenesNew England BiolabsCatalog #E3322S
ReagentCas9 Nuclease S. pyogenesNew England BiolabsCatalog #M0386S
ReagentMonarch® RNA Cleanup KitNew England BiolabsCatalog #T2050S
ReagentEnGen® sgRNA Synthesis Kit S. pyogenesNew England BiolabsCatalog #E3322S
ReagentCas9 Nuclease S. pyogenesNew England BiolabsCatalog #M0386S
ReagentMonarch® RNA Cleanup KitNew England BiolabsCatalog #T2050S
Designing oligos for gRNA synthesis
Designing oligos for gRNA synthesis
40m
40m
Use ChopChop tool (chopchop.rc.fas.harvard.edu) to select the gRNA of interest. Aurantiochytrium limacinum genome is available on ChopChop, and the gene of interest can be accessed using the gene co-ordinates of the JGI assembly https://mycocosm.jgi.doe.gov/Aurli1/Aurli1.home.html(For example: scaffold_10:1222284-1223740 (+)).
10m
Computational step
The output will list the gRNAs in a ranked order, considering the self-complementarity of the gRNA, efficiency, and off-target effects. Select the highly ranked gRNA and proceed for oligo designing for in vitro transcription.
10m

Note
Design target specific oligos following the NEB protocol

Select 20 nt gRNA sequences using the ChopChop web tool (do not include the PAM sequences), and add 'G' to the 5' end of the sequence, only if there is no 'G' at the 5' end.
10m
To the 5´ end; append T7 promoter sequence: TTCTAATACGACTCACTATA
To the 3´ end; append 14 nucleotide overlap sequence: GTTTTAGAGCTAGA
The final oligo sequence should look like 5´ TTCTAATACGACTCACTATAG(N)20GTTTTAGAGCTAGA 3´, (N)20 is the gRNA sequence.
This is the sequence of the oligo to be ordered.

Note
IMPORTANT: While the oligos are shipped, we recommend starting the pre-culture (step8) at this stage since it takes 2-3 days to grow.

10m
gRNA synthesis and purification
gRNA synthesis and purification
1h 30m
1h 30m


Note
Wear gloves and use nuclease-free tubes and reagents. Reactions should be assembled in microfuge tubes or PCR strip tubes.

Perform gRNA synthesis following the EnGen®sgRNA Synthesis Kit.

ReagentEnGen® sgRNA Synthesis Kit S. pyogenesNew England BiolabsCatalog #E3322S

1h
Critical
Purify the synthesized gRNA using Monarch RNA Cleanup Kit (50 μg)

ReagentMonarch® RNA Cleanup KitNew England BiolabsCatalog #T2050S


Quantify the gRNA using UV-Vis Nano-spectrometer.
Label and store the gRNA at Temperature-20 °C for short term storage and at Temperature-80 °C for long term storage.

30m
Ribonucleoprotein (RNP) preparation
Ribonucleoprotein (RNP) preparation
30m
30m
Thaw the gRNA and 1x PBS on ice. Spin down all the reagents before using.

For a total reaction volume of 5μl,
  1. Add Concentration120 pmol (final) gRNA and Concentration104 pmol (final) Cas9 protein.
  2. Adjust the reaction volume to 5μl using 1x PBS.
ReagentCas9 Nuclease S. pyogenesNew England BiolabsCatalog #M0386S
Mix the reagents by gently pipetting up and down. After a brief spin, incubate the tube at TemperatureRoom temperature for Duration00:20:00 .

Label and store the RNP (Cas9/gRNA) complex in Temperature-20 °C until further use.
Note


30m
Critical
Grow cells
Grow cells
4d
4d
  1. Start a pre-culture Duration96:00:00 (4 days) prior to electroporation by inoculating Amount5 mL of GPY (0.5% Yeast Extract, 1% Peptone, 3% D+-Glucose, 1.8% instant ocean) inAmount15 mL tube with a colony of Aurantiochytrium limacinum (ATCC MYA-1381). Incubate DurationOvernight at Temperature28 °C .
  2. Use preculture to inoculate Amount46 mL of GPY in Amount250 mL flask. Culture for 3 days at Temperature28 °C , Centrifigation171 rpm .


4d
Prepare reagents for Electroporation
Prepare reagents for Electroporation
2h
2h
  1. Prepare GPYs media (3% glucose, 0.6% peptone, 0.2% yeast extract, Concentration50 millimolar (mM) sucrose, 1.8% instant ocean)
  2. Prepare GPYs ampiciln (100μg/ml) plates (2%agar)
  3. Prepare GPYs zeocin (100μg/ml) and ampiciln (100μg/ml) plates (2%agar)
Note
GPYs zeocin + ampicillin plates are used if homology directed repair (HDR) template with ZeoR is used.

  1. Prepare 1X BSS (Concentration10 millimolar (mM) KCl, Concentration10 millimolar (mM) NaCl, and Concentration3 millimolar (mM) CaCl2)
  2. Prepare Concentration50 millimolar (mM) sucrose solution
  3. Sterilize by autoclave or filter sterilization as appropriate.

CITATION
Mariana Rius, Jackie Collier. Electroporation of Aurantiochytrium limacinum (ATCC MYA-1381).
LINK
dx.doi.org/10.17504/protocols.io.qjcduiw

2h
Prepare cells for electroporation
Prepare cells for electroporation
45m
45m
  1. Count cells using haemocytometer. Cell density should be around ~ 5 x10^7 cells/ml.
  2. Add 1.5 ml of cells to a microcentrifuge tube for each electroporation reaction. Centrifuge
  3. Centrifigation11000 rpm, 4°C, 00:05:00
  4. Decant the supernatant. Add 500 ul chilled 1X BSS (Concentration10 millimolar (mM) KCl, Concentration10 millimolar (mM) NaCl, and Concentration3 millimolar (mM) CaCl2). Centrifuge Centrifigation11000 rpm, 4°C, 00:05:00
  5. Decant the supernatant. Add Amount500 µL chilled Concentration50 millimolar (mM) sucrose. Centrifuge Centrifigation11000 rpm, 4°C, 00:05:00 . Repeat thrice (3x).
  6. Re-suspend by scraping cell mass off side of tube and vortex.

45m
Add RNP
Add RNP
5m
5m
  1. Add Amount5 µL of RNP complex mixture to suspended cells.
  2. Incubate on ice Duration00:05:00 .
  3. Transfer to chilled electroporation cuvette (0.2cm; BioRad).
  4. Keep the cuvettes on ice.
  5. Keep appropriate negative control using the same volume of elution buffer or water.
5m
Electroporation
Electroporation
10m
10m


Equipment
NEPA21 Super Electroporator
NAME
NEPAGENE
BRAND
NEPA21
SKU
http://www.nepagene.jp/e_products_nepagene_0001.html
LINK

Clean the loading pedestal and the cuvette before loading.

Set the parameters for poring and transfer pulse,
Poring pulse:

Voltage (V)Pulse length (ms)Length interval (ms)No. of pulseDecay rate (%)Polarity
275850210+

Transfer pulse:
Voltage (V)Pulse length (ms)Length interval (ms)No. of pulseDecay rate (%)Polarity
205050140+/-

The capacitance and resistance is set at 125 μF, and 1000 Ω respectively.

Pulse the cells and record the time constant. Take out the cuvette and place it on ice. Repeat the steps for the rest of the samples.
10m
Critical
Outgrowth and plating
Outgrowth and plating
1h 30m
1h 30m
  1. Add 1ml GPYs media to the cuvette and carefully aspirate out the cells on to a fresh micro centrifuge tube.
  2. Label the corresponding tubes and Incubate at Temperature28 °C for Duration01:00:00 without shaking.
  3. Centrifuge the cells at Centrifigation11000 rpm, 25°C, 00:05:00 , and discard the supernatant.
  4. Re-suspend the cells in remaining supernatant by pipetting up and down and also by vortexing.
  5. Dilute the cells appropriately (100x, 1000x) in Amount100 µL of fresh GPYs media.
  6. Plate the cells on appropriate GPYs plates, we usually include ampicillin to control bacterial contamination, and incubate at Temperature28 °C .
1h 30m
Monitor transformant colonies
Monitor transformant colonies
2d
2d
Colonies of Aurantiochytrium limacinum transformants will appear 2 days after plating. Streak individual colonies on fresh GPYs plate, and observe the phenotype/genotype. Include wild type (WT) for comparison.

Note
The transformant selection/screening can vary depending on the target gene and the selection marker used. In our case, we knocked-out carotenogenic gene, and the phenotype selection was based on colony colour.

2d
Citations
Step 9
Mariana Rius, Jackie Collier. Electroporation of Aurantiochytrium limacinum (ATCC MYA-1381)
dx.doi.org/10.17504/protocols.io.qjcduiw