CRISPR/Cas9 Flag knockin

Knock-in of Flag into endogenous loci

Preparation of sgRNA expression plasmid and donor DNA
 

1. Design sgRNAs and donor DNA according to Richardson et al., Nature Biotech. 2015.
Optimal donor DNA is complementary to the non-target strand of the sgRNA and overlaps the Cas9 cut site with 36 bp on the PAM-distal side and with 91 bp on the PAM-proximal side.
2. Purchase donor DNA as ssDNA from commercial source.
3. Clone sgRNA into suitable expression plasmid (e.g. Plasmid PX459; Addgene # 62988).
 

Cloning procedure

1. Annealing single strand oligos    
1.1 Dissolve lyophilized oligos:   
Short spin at max speed in centrifuge to collect pellet   
Add appropriate amount of sterile water for a final concentration of 100uM 
Heat tube for 5min at 65°C   
Vortex for 10sec and spin down   
Let oligos return to RT   
1.2 For each pair of reverse and forward oligos, pipette the following reaction into a screw cap tube:   
10 µl oligo forward 
10 µl oligo reverse  
10 µl Tango Buffer (or similar)  
70 µl H₂O  
1.3 Bring to 95°C for 5min.   
1.4 Let oligos return to RT for 1-2hrs   
 
2. Ligation of double-stranded oligos into PX459 vector      
2.1 Set up the following Ligation reaction:   
150 ng of PX459 plasmid  
1 µl of double-stranded oligos  
2µl NEB T4 ligase buffer  
1 µl BbsI  
1 µl T4 ligase 
Water up to 20µl   
2.2 Run the following Ligation reaction in a thermocycler:   
                     Cycles
37°C   5 min   1
16°C   10 min   10 cycles (can be extended to 50 cycles overnight)
37°C   15 min   1
80°C   5 min    1
   
2.3 Transform Competent Bacteria with Ligation Reaction   

Knock-in procedure

Day 1
4. Plate cells of interest in 10 cm dish for transfection the next day. Cell density should reach 70-80% the next day.

Day 2
5. Transfect cells with knock-in constructs using a suitable transfection reagent (here: JetPrime).

   a. Mix the following components and vortex for 10 s

   b. Incubate for 10 min at RT
   c. Add 10 ml fresh medium to cells in the 10 cm dish
   d. Add transfection mixture dropwise to the cells
   e. Incubate cells over night
 

Day 3
6. Wash cells and add fresh medium.
 

Day 4
7. Split cells and plate some of the cells for immunofluorescence in suitable culture ware (e.g. chamber slides). Culture rest of the cells in a 10 cm dish.
 

Day 5-6
8. Perform immunofluorescence for detection of Flag in chamber slide (e.g. with Flag M2 mouse anti-Flag Ab).
9. Estimate fraction of positive cells.
 

Day 6
10. Transfer cells from 10 cm dish into 96 well plate. Use fraction of positive cells to calculate number of cells necessary to get an enrichment (e.g. plate cells necessary to get 10 positive wells per plate (=10 fold enrichment)).
 

Day 8-10
11. Duplicate 96 well plate into 2 new 96 well plates.
 

Day 12-13
12. Use one of the 96 well plates for detection of Flag by immunofluorescence.
13. Identify wells with highest number of positive cells
14. Grow up cells from other 96 well plate. Repeat enrichment step if necessary.