AAVS1 Knock-in

One day before nucleofection, prepare two DR4 MEFs 6-well plates.

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.

hPSCs Medium

A	B
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 µl
Heat Stable Recombinant Human FGF2 (25µg/ml)*	80 µl

*While we prefer Heat Stable Recombinant Human FGF2, we also have used regular FGF2. Final volume: 500ml

L-Glutamine (100X)

A	B
L-Glutamine, powder	14.6 g
MilliQ H2O	500 ml

2-Mercaptoethanol (10,000X)

A	B
2-Mercaptoethanol	0.78 ml
MilliQ H2O	9.22 ml

Heat Stable Recombinant Human FGF2 (25µg/ml)

A	B
Heat Stable Recombinant Human FGF2	500 µg
0.1% BSA	20 ml

Final volume: 20ml

Y-27632 (1,000X)

A	B
Y-27632	5 mg
DMSO	1.56 ml

hPSCs Medium + Rock Inhibitor

A	B
hPSCs medium	500 ml
Y-27632 (1,000X)	500 µl

Final volume: 500ml

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.

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

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

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

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

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.

Primer Sequences & Product Size

A	B	C
SP-AAVS1-HR-L	CCCGCTTCAGTGACAACGTC	1313bp
ASP-AAVS1-HR-L	GAACTCTGCCCTCTAACGCT
SP-AAVS1-HR-R	TGCATCGCATTGTCTGAGTAG	1184bp
ASP-AAVS1-HR-R	TACCCCGAAGAGTGAGTTTGC

PCR with GXL DNA polymerase

PCR with GXL DNA polymerase - Setup

A	B
Ultrapure H2O	11 µl
5x GXL buffer	4 µl
2.5 mM dNTP	1.6 µl
10 µM primer Forward	0.5 µl
10 µM primer Reverse	0.5 µl
PrimeStar GXL DNA polymerase	0.4 µl
Crude cell lysis	2 µl

Touch-down PCR program

Touch-down PCR program

A	B
98°C	3 min
98°C	30 s
70°C (touch down, 1C/cycle)	30 s
72°C	1 min
Go to 2	12 cycles in total
98°C	30 s
58°C	1 min
72°C	30 s
Go to 6	23 cycles in total
72°C	7 min
4°C or 12°C	forever

Run PCR products in agarose gels.

Gel purify the bands of positively targeted clones and perform sanger sequencing to confirm.

Thaw and expand the correctly targeted clones

Test clones for mycoplasma, stain for pluripotent markers, and karyotype