TITLE: CRISPR/Cas9 based knockout generation in Aurantiochytrium limacinum (ATCC MYA-1381)
AUTHORS: Anbarasu Karthikaichamy, Jackie Collier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Transformation protocol for electroporation of CRISPR/Cas9 ribonucleoprotein (RNP) complex in urantiochytrium limacinum (ATCC MYA-1381; Stramenopile/ Heterokont, Thraustochytrid). </div></div>

[STEPS]
?. [Grow cells]
Start a pre-culture   (4 days) prior to electroporation by inoculating  of GPY (0.5% Yeast Extract, 1% Peptone, 3% D+-Glucose, 1.8% instant ocean) in tube with a colony of Aurantiochytrium limacinum (ATCC MYA-1381). Incubate  at .Use preculture to inoculate  of GPY in  flask. Culture for 3 days at , .
5 ml
15 ml
28 °C
46 ml
250 ml
28 °C
Centrifuge: 171 33
?. [Prepare reagents for Electroporation]
Prepare GPYs media (3% glucose, 0.6% peptone, 0.2% yeast extract,  sucrose, 1.8% instant ocean)Prepare GPYs  ampiciln (100μg/ml) plates (2%agar)Prepare GPYs zeocin (100μg/ml) and ampiciln (100μg/ml) plates (2%agar) Prepare 1X BSS (  KCl,   NaCl, and   CaCl2)Prepare   sucrose solution Sterilize by autoclave or filter sterilization as appropriate.Prepare 1X BSS ( KCl,  NaCl, and  CaCl2)Prepare  sucrose solution
GPYs zeocin + ampicillin plates are used if homology directed repair (HDR) template with ZeoR is used.
?. [Designing oligos for gRNA synthesis]
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 (+)).
?. [Designing oligos for gRNA synthesis]
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.
?. [Designing oligos for gRNA synthesis]
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.
Design target specific oligos following the NEB protocol
?. [Designing oligos for gRNA synthesis]
To the 5´ end; append T7 promoter sequence: TTCTAATACGACTCACTATATo the 3´ end; append 14 nucleotide overlap sequence: GTTTTAGAGCTAGAThe 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.
IMPORTANT: While the oligos are shipped, we recommend starting the pre-culture (step8) at this stage since it takes 2-3 days to grow.
?. [gRNA synthesis and purification]
Perform gRNA synthesis following the EnGen®sgRNA Synthesis Kit.
Wear gloves and use nuclease-free tubes and reagents. Reactions should be assembled in microfuge tubes or PCR strip tubes.
?. [Ribonucleoprotein (RNP) preparation]
Thaw the gRNA and 1x PBS on ice. Spin down all the reagents before using.For a total reaction volume of 5μl, Add  (final) gRNA and  (final) Cas9 protein.Mix the reagents by gently pipetting up and down. After a brief spin, incubate the tube at  for  .Label and store the RNP (Cas9/gRNA) complex in  until further use.
0 Room temperature
-20 °C
OptionalCheck the efficiency of RNP by performing an in vitro cleavage assay.
?. [gRNA synthesis and purification]
Purify the synthesized gRNA using Monarch RNA Cleanup Kit (50 μg)Quantify the gRNA using UV-Vis Nano-spectrometer.Label and store the gRNA at  for short term storage and at  for long term storage.
-20 °C
-80 °C
?. [Prepare cells for electroporation]
Count cells using haemocytometer. Cell density should be around ~ 5 x10^7 cells/ml. Add 1.5 ml of cells to a microcentrifuge tube for each electroporation reaction. Centrifuge   Decant the supernatant. Add 500 ul chilled 1X BSS (  KCl,   NaCl, and   CaCl2). Centrifuge    Decant the supernatant. Add   chilled   sucrose. Centrifuge   . Repeat thrice (3x).Re-suspend by scraping cell mass off side of tube and vortex.Decant the supernatant. Add 500 ul chilled 1X BSS ( KCl,  NaCl, and  CaCl2). Centrifuge Decant the supernatant. Add  chilled  sucrose. Centrifuge  . Repeat thrice (3x).
Centrifuge: 11000 33, 5 min, 4 10
Centrifuge: 11000 33, 5 min, 4 10
500 µl
Centrifuge: 11000 33, 5 min, 4 10
?. [Add RNP]
Add  of RNP complex mixture to suspended cells. Incubate on ice . Transfer to chilled electroporation cuvette (0.2cm; BioRad). Keep the cuvettes on ice.Keep appropriate negative control using the same volume of elution buffer or water.
5 µl
?. [Electroporation]
Clean the loading pedestal and the cuvette before loading. Set the parameters for poring and transfer pulse,Poring pulse:  ABCDEF1Voltage (V)Pulse length (ms)Length interval (ms)No. of pulseDecay rate (%)Polarity2275850210+Transfer pulse:  ABCDEF1Voltage (V)Pulse length (ms)Length interval (ms)No. of pulseDecay rate (%)Polarity2205050140+/-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.
ABCDEF1Voltage (V)Pulse length (ms)Length interval (ms)No. of pulseDecay rate (%)Polarity2275850210+
ABCDEF1Voltage (V)Pulse length (ms)Length interval (ms)No. of pulseDecay rate (%)Polarity2205050140+/-
?. [Outgrowth and plating]
Add 1ml GPYs media to the cuvette and carefully aspirate out the cells on to a fresh micro centrifuge tube. Label the corresponding tubes and Incubate at    for    without shaking. Centrifuge the cells at   , and discard the supernatant.Re-suspend the cells in remaining supernatant by pipetting up and down and also by vortexing. Label the corresponding tubes and Incubate at   for   without shaking. Centrifuge the cells at  , and discard the supernatant.Dilute the cells appropriately (100x, 1000x) in  of fresh GPYs media.Plate the cells on appropriate GPYs plates, we usually include ampicillin to control bacterial contamination, and incubate at  .
28 °C
Centrifuge: 11000 33, 5 min, 25 10
100 µl
28 °C
?. [Monitor transformant colonies]
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.
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.