TITLE: YAP1 mKate:BSD HDR Knock-in (via Cas9 RNP lipofection)
AUTHORS: Emir  Bora Akmeriç

[DESCRIPTION]
s

[STEPS]
SECTION: Culturing Immortalized HUVECs
1. In a t25 flask, add 10 mL of EGM2 (with antibiotics). Incubate flask for 20 minutes

SECTION: Culturing Immortalized HUVECs
1.1. Thaw a 500k/1M CI-huVEC tube in a 37C water bath
and add the cells into the flask.

SECTION: Culturing Immortalized HUVECs
1.2. Change media 6-8 hours later or when cells have adhered to the flask surface to remove excess DMSO.
SECTION: Culturing Immortalized HUVECs
1.3. Culture until confluent. Prior to 80% confluency, change media every other day. After 80% confluency, change media daily.
SECTION: RNP Complex Formation and Reverse RNP Transfection
3. Prepare 10 μM eSpCas9 in HEPES buffer. For example:  7.32 μL of 41μM eSpCas9 in 22.86 HEPES buffer to have a final Volume of 30μL 10μM eSpCas9.

Prepare a working solution of 1 μM eSpCas9 in Opti- MEM™. 
Always prepare fresh Cas9 nuclease V3 working solution before complexing with crRNA:tracrRNA. 
SECTION: RNP Complex Formation and Reverse RNP Transfection
4. Mix 6 μL of crRNA:tracrRNA complex (1 μM), 6 μL of Cas9 working solution (1 μM), and 88 μL of Opti-MEM™ I per replicate to form crRNA:tracrRNA:Cas9 complexes in a tube. Incubate at room temperature for 5 min. Mix the protein solutions gently by inverting the tube.

For exapmle for 5 wells in 24 well plate
30μL of crRNA:tracrRNA complex (1 μM)
440μL of Opti-MEM™
SECTION: RNP Complex Formation and Reverse RNP Transfection
5. Combine 100 μL of the crRNA:tracrRNA:Cas9 complexes , 4.8 μL of Lipofectamine™ RNAiMAX, and 95.2 μL of Opti-MEM™ I per replicate to get the final transfection solution. Incubate at room temperature for 20 min. Mix properly by pipetting up and down or by inverting the tube.

SECTION: RNP Complex Formation and Reverse RNP Transfection
6. Wash CI-huVEC with PBS during incubation of the transfection solution. Trypsinize the cells, stop the reaction with FBS and EBM 1:1:1, after detachment and centrifuge for 5 min at 300 x g. Resuspend the cell pellet in EGM2 ( -Gentamycin) and count the cells.
SECTION: RNP Complex Formation and Reverse RNP Transfection
7. Use EGM2(-Gentamycin) to adjust the cell concentration to 90,000 cells per mL.

SECTION: RNP Complex Formation and Reverse RNP Transfection
8. After incubation of the transfection solution, pipette 200 μL of the transfection solution into one well of a 24-well plate. Gently mix the complexes by pipetting before adding to the well.
SECTION: RNP Complex Formation and Reverse RNP Transfection
9. Add 400 μL of the cell suspension to the transfection complexes to obtain a final concentration of 10 nM RNP and 36,000 cells/well. Gently mix by pipetting and swirl the plate to ensure sufficient distribution of cells and complexes
SECTION: RNP Complex Formation and Reverse RNP Transfection
10. Incubate at 37 °C and 5% CO2. Change medium ( EGM2) every 2–3 days.
SECTION: RNP Complex Formation and Reverse RNP Transfection
11. When cells reach at least 80% confluency, wash with 500 μLof PBS and trypsinize with 200 μL of Trypsin/EDTA. Stop the reaction with 1 mL of EGM2 after cell detachment.

SECTION: RNP Complex Formation and Reverse RNP Transfection
12. Subculture 800 μL (2/3 of the cells) to one well of a 6-well plate, add 1 mL EGM2, and culture the cells at 37 °C and 5% CO2 . 
SECTION: RNP Complex Formation and Reverse RNP Transfection
13. Centrifuge the remaining cells (1/3) for 5 min at 300 x g. Aspirate and discard the supernatant. The supernatant should be removed completely, as any residual medium can inhibit PCR efficiency. Resuspend and lyse the cell pellet in 50 μL of  our DNA Extraction Solution and transfer the lysate to a PCR tube.

SECTION: Estimation of Genome Editing  Efficiency
14. Materials for Estimation of Genome Editing  Efficiency:
1.  or NEBuffer™2 10x (NEB): 500mMNaCl, 100mMTris–HCl, 100 mM MgCl2, 10 mM DTT, pH 7
2. Nuclease-free H2O
3.    
4. Agarose gel

5.Gel loading buffer (6x)

6. TBE Buffer 10x: 890 mM Tris, 890 mM boric acid, 20 mM Titriplex III




Incubate extracted DNA at 65 °C for 15 min and for another 15 min at 95 °C in a thermal cycler. Store at 4 °C.
SECTION: Estimation of Genome Editing  Efficiency
15. Determine the DNA concentration of the lysed cells DNA extract at the Nanodrop.

SECTION: Estimation of Genome Editing  Efficiency
16. Amplify the target region by PCR using 2–20 ng of genomic template DNA regarding the PCR protocol. 
SECTION: Estimation of Genome Editing  Efficiency
17. Following PCR, form heteroduplexes by combining 5 μLof PCR product with 1.5 μL of NEBuffer™ 2 (10x) and 6.5 μL of nuclease-free H2O. Use 1.5 μL of T7EI (10 U/μL) and NEBuffer™2 (10x) stock solutions and fill up to 15 μL with nuclease-free H2O to dilute T7EI to 1 U/μL. Volumes can be adjusted as required

SECTION: Estimation of Genome Editing  Efficiency
18. Heat to 95 °C in a thermal cycler for 10 min and cool down to 85 °C with a ramp rate of -2 °C/s. Next, cool down to 25 °C with a ramp rate of -0.3 °C/s.
SECTION: Estimation of Genome Editing  Efficiency
19. Prepare a 2% agarose gel with 1x TBE in a gel tray. 
SECTION: Estimation of Genome Editing  Efficiency
20. Add 3 μL of 6x loading dye and separate the digested products  by gel electrophoresis. A unique band pattern indicates successful CRISPR/Cas9 genome editing.

During T7EI digestion, mismatch positions in the hetero duplexed PCR products are specifically cleaved, whereas homo- duplex DNA will not. This results in three specific bands after separation by gel electrophoresis. Measuring their intensities enables an estimation of the CRISPR/Cas9-induced indel rate.
 

SECTION: Estimation of Genome Editing  Efficiency
21. Determine the volume intensities of the fragments and estimate indel rates as described in Ran FA et al before.


  

SECTION: Estimation of Genome Editing  Efficiency
22. 
SECTION: RNP Complex Formation and Reverse RNP Transfection
2.2. For each condition: 
Mix 1 μL of each stock solution (crRNA and tracrRNA) with 98 μL nuclease-free duplex buffer to form 1 μM crRNA:
tracrRNA complexes. Incubate at 95 C for 5 min and allow the complexes to cool down to room temperature

SECTION: RNP Complex Formation and Reverse RNP Transfection
2.1. Resuspend lyophilized crRNA and tracrRNA in nuclease-free TE buffer to stock concentrations of 100 μM.
SECTION: RNP Complex Formation and Reverse RNP Transfection
2. Materials for Reverse RNP Transfection:
1.  
2.   
3.   or  
4. Alt-R® CRISPR-Cas9 crRNA (Integrated DNA Technologies [IDT]  for YAP1 (HDR_YAP_Pref 5'- /AltR1/rUrUrA rGrArA rUrUrC rArGrU rCrUrG rCrCrU rGrArG rUrUrU rUrArG rArGrC rUrArU rGrCrU /AltR2/ -3')  )

5.CRISPR-Cas9 tracrRNA (IDT)

6. MDC Homebrew eSpCas9 in HEPES buffer(pH 7.5)(41 µM)
6b. 20 mM Hepes pH 7.5, 0.15 M KCl, 1 mM DTT

7. As negative CRISPR/Cas control: Alt-R® CRISPR-Cas9 Negative Control crRNA #1