8. The transfection efficiency of my cell line is only 20%, how do I enrich CRISPR transfected cells?

You can use pCas-Guide-EF1a-GFP to enrich transfected cells since GFP is also expressed. We also have pCas-Guide-EF1a-CD4 vector; you can use anti-CD4 antibody beads to enrich transfected cells. Alternatively, you can transfect a plasmid with a selection marker and select the cells. Lenti vector can be used and integration-deficient lentivirus can be produced using the special integration-deficient lenti packaging kit (cat# TR30036); the lenti CRISPR vectors can be delivered into hard-to-transfect cells, but not integrating into the host genome.

11. How to screen the edited cells after transfecting the CRISPR/Cas9 vector?

For mutations, you can do genomic PCR and sequence it. If you do gene knockout, the selection marker in the donor template DNA will help the selection. If no donor DNA for gene knockout out, then genomic PCR and sequencing to confirm indels. If necessary, you can isolate individual cell colonies for introduction of specific mutations and other genome editing applications. You can do WB for gene knockout after isolating single cell colonies. 

21. What is the known CRISPR/Cas9 editing efficiency relative to other genome editing approaches?

In general, the genome editing efficiency of CRISPR/Cas9 is similar or higher than TALEN. However, CRISPR/Cas9 is much more simple and easy to do. You will need to engineer the protein to recognize new DNA sequence in TALEN system, while CRISPR/Cas9 is RNA based.

To provide convenience to you, we offer a target sequence cloning service into any of OriGene’s CRISPR/Cas vectors. With a turn around time of only 7-10 days, we are offer a competitive service. Click the banner below to request a free quote!