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

[DESCRIPTION]
s

[STEPS]
SECTION: RNP Complex Formation and Reverse RNP Transfection
3.  Resuspend lyophilized crRNA and tracrRNA in nuclease-free TE buffer to stock concentrations of 100 μM.
SECTION: RNP Complex Formation and Reverse RNP Transfection
5. To form crRNA:tracrRNA:Cas9 complexes in a tube and to start excision of homology casette, mix 6 μL of crRNA:tracrRNA complex (1 μM), 6 μL of Cas9 working solution (1 μM), and HDR Donor Plasmid(0.6-1.5 µg). Bring volume to 100 uL using OptiMEM. Mix the protein solutions gently by inverting the tube.

For example for 5 wells in 24 well plate
1)30μL of crRNA:tracrRNA complex (1 μM)
2)10 nM-25 nM HDR Donor Plasmid(Set two replicates with two concentrations per condition)
3)30μL of Cas9 (1 μM)
4)OptiMEM to bring to 500 µL
SECTION: RNP Complex Formation and Reverse RNP Transfection
6. Combine both tubes 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
7. Wash CI-huVEC with warm 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 500 x g. Resuspend the cell pellet in EGM2 ( -Gentamycin) and count the cells.
SECTION: RNP Complex Formation and Reverse RNP Transfection
8. Use EGM2(-Gentamycin) to adjust the cell concentration to 90,000 cells per mL.

SECTION: RNP Complex Formation and Reverse RNP Transfection
9. 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
10. 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
11. Incubate at 37 °C and 5% CO2. 
SECTION: RNP Complex Formation and Reverse RNP Transfection
12. When cells reach at least 80% confluency, wash with 500 μLof PBS and trypsinize with 200 μL of Trypsin/EDTA. Stop the reaction with 0.5 mL EGM2+0.5 mL FBS after cell detachment.

SECTION: RNP Complex Formation and Reverse RNP Transfection
13. 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
14. 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
15. Determine the DNA concentration of the lysed cells DNA extract at the Nanodrop. Either do steps 16-21 OR do PCR and gel staining using primers for Genomic PCR primers. For the latter approach, have Genomic PCR primers and untreated cell culture DNA extract ready.

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: Culturing Immortalized HUVECs
1.2. Culture until confluent. Prior to 80% confluency, change media every other day. After 80% confluency, change media daily.
SECTION: Culturing Immortalized HUVECs
1.1. Change media 6-8 hours later or when cells have adhered to the flask surface to remove excess DMSO.
SECTION: Culturing Immortalized HUVECs
1. Thaw a 500k/1M CI-huVEC tube in a 37C water bath
and add the cells into the flask.

SECTION: RNP Complex Formation and Reverse RNP Transfection
3.2.  Heat the mixture at 95C for 5 minutes, followed by letting it cool to room temperature on the bench top.
SECTION: RNP Complex Formation and Reverse RNP Transfection
4. 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. Mix well and briefly centrifuge


SECTION: RNP Complex Formation and Reverse RNP Transfection
3.1. To prepare the gRNA complex, combine the crRNA and tracrRNA. Take 1 uL from each component, and add 98 µL of Duplex Buffer to bring it to a final concentration of 1 µM.

SECTION: RNP Complex Formation and Reverse RNP Transfection
4.1. Prepare a working solution of 1 μM eSpCas9 in Opti- MEM™. 
Always prepare fresh Cas9 nuclease rking solution before complexing with crRNA:tracrRNA.
SECTION: RNP Complex Formation and Reverse RNP Transfection
5.1. In a seperate tube, per well, mix 4.8 uL of Lipofectamine RNAiMAX and 95.2 µL Opti-MEM. Incubate for 5 minutes
SECTION: RNP Complex Formation and Reverse RNP Transfection
8.1. If IDT HDR Enhancer  V2 is used, add 1 µM HDR Enhancer V2(Stock concentration 690 µM) to the mixture.
IMPORTANT: IF ENHANCER IS ADDED, MAKE A CONTROL SAMPLE WITH 1% DMSO
SECTION: RNP Complex Formation and Reverse RNP Transfection
11.1. After 24h change medium  w/ EGM2 without antibiotics.
SECTION: RNP Complex Formation and Reverse RNP Transfection
11.2. 48 hours post transfection, change medium w/ EGM2 with Blasticidin S. 10 µg/mL concentration is suggested per Anna's experiments for immortalized HUVECs and HUVECs in general. Change media depending on overall cell status preferably after 48 hours.
SECTION: RNP Complex Formation and Reverse RNP Transfection
11.3. 3-4 days after antibiotic addition, check the positive conditions on the 24 well plate under a light microscope for cell fitness, morphology and overall phenotype. Additionally, check for mKate2 fluorescence and acquire images of ~200 cell in total. From these conditions, mark the wells you would like to subculture.

NOTICE:  If the fluorescent cell ratio is above 90%, continue with the expansion(step 12). For fluorescence ratio between 10-90%, use a dilution assay to produce single cell colonies or FACS sorting(different protocol).
SECTION: RNP Complex Formation and Reverse RNP Transfection
2. Make a table for set of conditions on a 24 well plate-
SECTION: Estimation of Genome Editing  Efficiency
15.1. Following gel assay, send two components for sequencing:
1) DNA extract
2)Sequencing primer for YAP Exon 9 C-termini. The sequencing, if successful, would provide ab1 files with nucleotide sequences of Donor plasmid casette
SECTION: Immunofluorescence/Live Microscopy Testing
22.