TITLE: Protocol for introducing fluorescently labeled CRISPR/Cas9 RNP complex into heterotrophic dinoflagellates
AUTHORS: Brittany Sprecher, Huan Zhang, Senjie Lin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Introducing CRISPR/Cas9 machinery into the cell as RNP complexes reduces the number of steps necessary to edit the genome, does not require codon optimization or a strong promoter, can result in higher editing efficiencies with fewer off target effects, and has lower risks of cell toxicity (Woo et al., 2015; Liang et al., 2015; Liang et al., 2017; Farboud et al., 2018).  This protocol utilizes IDTs fluorescently labeled tracrRNAs to visually check to see whether the CRISPR/Cas9 RNP molecule can make it into non photosynthetic dinoflagellates.  This protocol serves as a good starting point to evaluate the CRISPR/Cas9 RNP method, the relative transformation efficiency, and can be used to help sort cells that contain the CRISPR/Cas9 RNP molecule from cells that do not.</div></div>

[STEPS]
?. [1.	Prepare the RNP complex from IDT]
Combine 1 uL Alt-R™ CRISPR/Cas9 tracrRNA – ATTO™ 550 (100 uM) + 1 L Alt-R™ CRISPR/Cas9 crRNA (100 uM) + 18 uL IDT Nuclease Free Buffer
?. [1.	Prepare the RNP complex from IDT]
Incubate at 95˚C for 5 minutes, let cool to room temperature on bench
?. [1.	Prepare the RNP complex from IDT]
Meanwhile, combine 2.67 uL Alt-R Cas9 Enzyme (61 uM) + 37.33 uL IDT Nuclease Free Buffer
?. [1.	Prepare the RNP complex from IDT]
Form RNP complex by combining 1-part gRNA (part 1) and 1-part Cas9 (part 3) and let incubate at room temperature for 20 mins.
?. [1.	Prepare the RNP complex from IDT]
Note: the concentrations can and should be adjusted to determine optimal [RNP] and Cas9:gRNA ratios
?. [1.	Prepare the RNP complex from IDT]
Note: RNP complexes can be kept at 4˚C for a month or 5 months at -80˚C
?. [Collecting O. marina]
Concentrate healthy Oxyrrhis marina cells using a flashlight. Make sure that under green light the cells have no background fluorescence (this may require not feeding them for some time)
?. [Collecting O. marina]
Count cell concentration of these healthy cultures using a Sedgewick Rafter Counting Chamber.  Goal is to use 200,000 cells/well for electroporation
?. [Collecting O. marina]
Spin down the O. marina cells that are necessary for the experimental set-up (need to include the NPC (no pulse controls) and scrambled gRNA control). Use 2500g for 2.5 mins
?. [Collecting O. marina]
Remove all but 2-5 mL of liquid, being very careful to not disturb the pellet. Transfer gently to 2 mL test tubes
?. [Collecting O. marina]
Centrifuge at 900g for 1 minute and remove all remaining liquid
?. [Collecting O. marina]
Resuspend in electroporation buffer
?. [Collecting O. marina]
For each experimental well use Lonza’s 16.4 uL SG Buffer, Lonza’s 3.6 uL Supplement 1 Solution, and 2uL RNP complex (Step 4)
?. [Electroporation Steps]
Place 22 uL into each well of Lonza’s Nucleocuvette™ 16-well strips very gently and electroporate using desired setting
?. [Electroporation Steps]
Immediately add 80 uL of filtered and autoclaved seawater containing the antibiotic cocktail to limit bacteria growth
?. [Electroporation Steps]
After 20-30 minutes transfer each well to a 24-well culture plate that already contains 1.4 mL of the same seawater described above
?. [Observation Steps]
After 1.5-2 hours try to observe the cells for red fluorescence to demonstrate RNP complex uptake under green light
?. [Observation Steps]
Continue to observe cells for fluorescence for 3 days (many experiments have shown RNP complexes are mostly degraded after 72 hours)
?. [Observation Steps]
Isolate gDNA after ~72 hours and perform T7E1 endonuclease assay to determine gene editing efficiency