TITLE: AAVS1 Knock-in
AUTHORS: Hanqin  Li, Dirk Hockemeyer

[DESCRIPTION]
This protocol describes the standard procedure to knock-in constructs to the AAVS1 safe harbor locus in hPSCs. 

General Notes:
1. The AAVS1 knock-in construct, AAVS1-SA-neo-CAGGS-PE2-2A-GFP, can be found at AddGene (Catalog: 180014, RRID:Addgene_180014)
2. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.


[STEPS]
1. One day before nucleofection, prepare two DR4 MEFs 6-well plates.
2. Nucleofection of Cas9/sgRNA RNP (protospacer sequence, ACCCCACAGTGGGGCCACTA) and AAVS1 knock-in targeting vector is performed using the nucleofection of ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) protocol as described in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs;" dx.doi.org/10.17504/protocols.io.b4qnqvve

After nucleofection, seed all cells onto two 6-well plates containing hPSCs medium + Rock Inhibitor. 


3. From day 3, change medium daily for 10 days with hPSCs medium with 70 µg/ml G418. Most of the hPSCs will die during the G418 selection.

4. When large hPSC colonies emerge, manually pick and re-plate them individually in 12-well ICR MEFs plates, as described in the collection "Standard operating procedure for the isolation of genetically engineered hPSCs clones in a high-throughput way;” dx.doi.org/10.17504/protocols.io.b4mmqu46
5. When these expanded clones grow to 50%, passage 1/4 to a new well of a 6-well plate for further expanding. 

For a detailed protocol on passaging hPSCs, refer to the collection "Thawing, Passaging and Freezing of hPSCs on MEFs;" dx.doi.org/10.17504/protocols.io.b4msqu6e
7. Freeze the expanded cells when they grow up.

For a detailed protocol on freezing hPSCs, refer to the collection "Thawing, Passaging and Freezing of hPSCs on MEFs;" dx.doi.org/10.17504/protocols.io.b4msqu6e
8. Genotype crude cell lysate from step 6 using the primers flanking each homologous arm with GXL DNA polymerase. Use unedited cells as a negative control.
9. PCR with GXL DNA polymerase

9.1. PCR with GXL DNA polymerase - Setup
 

10. Touch-down PCR program
10.1. Touch-down PCR program
 

11. Run PCR products in agarose gels.
12. Gel purify the bands of positively targeted clones and perform sanger sequencing to confirm.
13. Thaw and expand the correctly targeted clones

 
14. Test clones for mycoplasma, stain for pluripotent markers, and karyotype
8.1. Primer Sequences & Product Size
 

2.1. hPSCs Medium
     DMEM/F12      385 ml        Fetal Bovine   Serum (FBS)      75 ml        Knockout Serum Replacement      25 ml        L-Glutamine (100X)      5 ml        Penicillin & Streptomycin (100X)      5 ml        MEM Non-Essential Amino Acids (100X)      5 ml        2-Mercaptoethanol (10,000X)      50 ul        Heat Stable Recombinant Human FGF2 (25ug/ml)*      80 ul   
 
L-Glutamine (100X)
     L-Glutamine,   powder      14.6 g        MilliQ H2O      500 ml   
 
2-Mercaptoethanol (10,000X)
     2-Mercaptoethanol      0.78 ml        MilliQ H2O      9.22 ml   
 
Heat Stable Recombinant Human FGF2 (25µg/ml)
     Heat Stable Recombinant Human FGF2      500 ug        0.1% BSA      20 ml   
 
 Y-27632 (1,000X)
     Y-27632      5 mg        DMSO      1.56 ml   
 
 hPSCs Medium + Rock Inhibitor
     hESCs medium      500 ml        Y-27632 (1,000X)      500 ul   

6.  Prepare crude cell lysate from the rest of the cells for genotyping as described in the collection “Genotyping by next generation sequencing;" dx.doi.org/10.17504/protocols.io.b4n3qvgn