CRISPR/Cas9-mediated editing and cell sorting

CRISPR/Cas9-Mediated Homologous Recombination

Donor Plasmid Construction:

1. Linearize backbone plasmid pUC57 by double restriction-enzyme digestion.

2. PCR amplification of homologous arms from the HEK293FT cell DNA.
    Note: When designing primers, add 15-20 bp overlap sequences for later seamless cloning. The front homologous arm is a 501-bp fragment before the peptide stop codon and the back homologous arm is a 501-bp fragment starting with the peptide stop codon.

3. PCR amplification of the EGFP fragment from any plasmid containing this element.

    Note: When designing primers, add 15-20 bp overlap sequences for later seamless cloning, omit the start codon and the stop codon of the EGFP coding sequence.

4. Assemble the linearized vector and amplified fragments by a commercial seamless cloning kit. In our case, the NEBuilder® HiFi DNA Assembly Master Mix worked well.

5. Transform assembled plasmids into DH5α competent E. coli.

6. Pick single colonies for Sanger sequencing and retrieve the correct donor plasmids for further transfection.

Cloning sgRNA into the PX459-Cas9 Vector:

1. Pick a sgRNA targeting the genomic sequence near the peptide coding region using an online designer. In our case, we used CHOPCHOP.

2. Oligo annealing and cloning into PX459:

a. Digest 1 μg of PX459 with BbsI for 30 min at 37°C:

1 μg Plasmid

1 μl FastDigest BbsI

1 μl FastAP

2 μl 10× FastDigest Buffer

X μl ddH₂O

20 μl in total

b. Gel purify linearized plasmids.

c. Phosphorylate and anneal the sgRNA oligo pair:

1 μl oligo 1 (100μM)

1 μl oligo 2 (100μM)

1 μl 10× T4 Ligation Buffer

6.5 μl ddH₂O

0.5 μl T4 PNK

10 μl in total

Anneal in a thermocycler using the following parameters:

37°C 30 min

95°C 5 min and then ramp down to 25°C at 5°C/min

d. Set up a ligation reaction and incubate at room temperature for 10 min:

X μl linearized and purified plasmids from step b (50 ng)

1 μl phosphorylated and annealed oligo duplex from step c (1:200 dilution)

5 μl 2× Quickligation Buffer

X μl ddH₂O

1 μl Quick Ligase

11 μl in total

e. Transformation and plasmid retrieval for further transfection.

Edit the Genome and Analyze the Results

1. Plate 2 wells of HEK293FT cells at 3×10⁵/well in a 12-well cell culture plate.

2. Transfect cells with the donor plasmid and the PX459-sgRNA plasmid using Lipofectamine 3000 on the second day.

3. Two days after transfection, assess the EGFP expression by fluorescent microscopy or flow cytometry.

4. Index sort the EGFP positive cells and culture the cell clones.

5. PCR amplify the edited genomic region for Sanger sequencing.

Note: Design primers that span over the homology arms to avoid the amplification from the remaining donor plasmid if there is any.

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