TITLE: Step by step guide to tag  endogenous genes  with split-wrmScarlet and/or split-sfGFP in C. elegans
AUTHORS: Jerome F Goudeau, Cynthia Kenyon, Maria  Ingaramo

[DESCRIPTION]
Here is a step by step protocol describing our strategy to label endogenous proteins with split-sfGFP and/or split-wrmScarlet in C. elegans. See our manuscript "Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans " for more details.

[BEFORE_START]
If you are not already, get familiar with CRISPR/Cas genome editing, and general guidelines.

Kohler S, Dernburg A. (2016) C. elegans injection: Ribonucleoprotein delivery using the Alt-R CRISPR-Cas9 System. [Online] Coralville, Integrated DNA Technologies. [December, 2017.]
 

 

 





[GUIDELINES]
Here is a step by step protocol describing our strategy to label endogenous proteins with split-sfGFP and/or split-wrmScarlet in C. elegans. See our manuscript "Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans " for more details.

[STEPS]
SECTION: Select a C. elegans strain expressing the split-fluorescent protein1-10 of your choice
1. Select a C. elegans strain expressing wrmScarlet1-10 and/or sfGFP1-10 in your tissue of interest

Somatic split-sfGFP1-10:
CF4587 muIs253[(Peft-3::sfGFP1-10::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III

Somatic split-wrmScarlet1-10:
CF4582 muIs252[Peft-3::wrmScarlet1-10::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III

Dual Somatic split-sfGFP1-10 and split-wrmScarlet1-10:
CF4588 muIs253[Peft-3::sfGFP1-10::unc-54 3'UTR, Cbr-unc-119(+)], muIs252[Peft-3::wrmScarlet1-10::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III

Muscle-specific split-wrmScarlet1-10:
CF4610 muIs257[Pmyo-3::wrmScarlet1-10::unc-54 3'UTR] I
SECTION: Select a guide sequence and order a crRNA and tracrRNA
2. Download the DNA sequence of your gene and transcript of interest from Wormbase.
Identify the desired insertion or knock-in site in the genomic DNA.
SECTION: Select a guide sequence and order a crRNA and tracrRNA
3. Using ~50 nucleotides flanking Identify DNA sequence(s) followed by a PAM site (5' NGG 3') in your target gene using a CRISPR/Cas9 target online predictor, such as CCTop. 

When using CCTop, use the default parameters and adjust the following two criteria:
    PAM type: NGG (Streptococcus pyogenes)
    Species: C. elegans

Insert the DNA sequence in the query section, then submit.
Select the crRNA target site with a high score, the closest to your editing site, with no off- targets.
SECTION: Select a guide sequence and order a crRNA and tracrRNA
4. Order the crRNA corresponding to your selected guide sequence from IDT.
Note: Do not include the PAM in the query
Typically, we order 10 nmol of each Alt-R® CRISPR-Cas9 crRNA that we ressuspend in 14.5 μL TE.
SECTION: Select a guide sequence and order a crRNA and tracrRNA
5. Order the universal 67 mer tracrRNA from IDT under the section "CRISPR-Cas9 tracrRNA".
https://www.idtdna.com/pages/products/crispr-genome-editing/alt-r-crispr-cas9-system
Typically, we order 20 nmol.
SECTION: Design and order a single-stranded donor oligonucleotides (ssODN) of 200 mers
6. Design the sequence of a single-stranded donor oligonucleotides (ssODN) with the Fluorescent Protein11 of choice, (ie. wrmScarlet11 or sfGFP11), a linker and homology arms.

Sequences:
wrmScarlet11:
5' TACACCGTCGTCGAGCAATACGAGAAGTCCGTCGCCCGTCACTGCACCGGAGGA 3'

sfGFP11:
5' CGTGACCACATGGTCCTTCATGAGTATGTAAATGCTGCTGGGATTACA 3'

Linker:
5' GGAGGAGGATCC 3'

 

 

 

 

Order the ssODN from IDT in the section "Ultramer DNA Oligos". 
We typically order up to 4 nmol at 100 μM in IDTE buffer

SECTION: Design oligos to amplify the insertion site
7. Design and order oligos that will be used to perform the genotyping and sequencing of the insertion site.
 

SECTION: Prepare and inject CRISPR/Cas9 ribonucleoprotein mix
8. Assemble CRISPR/Cas9 ribonucleoproteins complex and ssODN into injection mix:

 

Alternatively:
 

Pipet up and down a few times without introducting air in the mix.
Incubate 37 °C 
Quick spin 13000 rpm 
Load 0.5 to 1 μL of the mix in injection needles and inject 10 to 20 day-1 adult worms successfully, ideally in both gonad arms.

Single injected worms in a drop of M9 buffer on NGM plates seeded with OP50, and place them in a 25 °C incubator  .
SECTION: Step F - Screen for successful FP11 integrants
9. Starting 3 days post injections, screen daily until identifying fluorescent progeny in the F1 and/or F2 of singled injected worms.
Perform PCR genotyping and sequencing using regular worm protocols with the primers designed in step C.