CRISPR Stable Knockin Cell Line Generation

* Choose from HEK293, HEK293T, A549, HeLa, MDCK, A375, HepG2, HT1080 or U87MG. These cell lines are available as Cas-9 expressing versions or parental version. Cas9 expressing version will be used by default.If the parental version is preferred, please request prior to order placement.
**This pricing is valid for a single gene knock-in for diploid loci only.
#Customer is required to provide at least 2 million cells, 1L of propagation media/any special coated flasks if needed (if cells need DMEM or RPMI, ABM will supply media). Also, the cell line must tolerate single cell cloning and display adequate transfection and transduction efficiency.
##Pre-validated antibody needs to be provided by customer with appropriate positive control (preferably with supporting data).
^All “add on” services need to be indicated prior to order placement as the project design will need to be finalized prior to start of the service.
^^ Final Lead time may vary depending on actual growth rate of cell line while expanding from single cell.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system offers cutting edge RNA-Guided endonuclease technology for highly specific and customizable genome editing. With the use of a target sequencespecific guide RNA, as well as the highly specific and programmable CRISPR associated nuclease (Cas9), abm offers an efficient and targeted genome editing service with minimal off-target effects.



CRISPR can be used to knock-in genes, tags, markers and more. With abm’s custom CRISPR Knock-In Service, you simply need to describe the desired knock-in and select a cell line, we do the rest.

Using CRISPR to Knock-in Red Fluorescent Protein (RFP) gene into Human Embryonic Kidney Cells at the AAVS1 Safe Harbour Site

• An expression cassette containing RFP and puromycin resistance genes (pAAVS1-RFP-DNR) was knocked into the AAVS1 Safe-harbor site in HEK293 cells using CRISPR targeted genome editing via the HDR pathway. Gene insertion at a Safe-harbour site allows stable gene expression without any adverse effects on the fitness of the engineered cells.
• Genomic PCR confirmed Knocked –in RFP integration at AAVS1 Safe-harbor locus.
• RFP expression was confirmed in cells by fluorescence microscopy.

Figure 1. CRISPR Knock-in requires expression of Cas9 and sgRNA to produce a double-stranded break. The repair template, shown here as pAAVS1-RFP-DNR, is used by the cell to repair the break using homologous recombination. The desired gene and selection marker (RFP and puromycin) included between the homology arms on the repair template will be integrated into the genome.

Phase #1: Construction and Delivery of sgRNA, Cas9 and Repair Template

• A sgRNA was designed against the human AAVS1 Safe-harbor locus.
• Software analysis was performed to ensure the sgRNA had no predicted off targets s. The selected sgRNA design, along with the CMV-promoter driven Cas9 gene, was cloned into pCas-Guide to make pCas-Guide-AAVS1 (Figure 2).
• The pAAVS1-RFP-DNR donor plasmid was designed to contain the RFP-puromycin expression cassette, flanked on either side by homology arms of 600 bp (Figure 2).
• HEK293 cells were co-transfected with both plasmids using DNAfectin transfection reagent.

Figure 2. Vector maps of pCas-Guide-AAVS1 and pAAVS1-RFP-DNR. pCas-Guide-AAVS1 is an all-in-one vector for co-expression of sgRNA and Cas9 in mammalian cells. Expression of sgRNA is driven by the U6 promoter, a strong constitutive Pol III promoter; while a CMV promoter drives the expression of the Cas9 enzyme. pAAVS1-RFP-DNR expresses puromycin resistance marker under the PGK promoter and RFP gene under the CMV promoter. The 5’ and 3’ AAVS1 homology arms (‘AAVS-Right’ and ‘AAVS-Left’) provide the cells with a template for Homology Directed Repair.


Phase #2: Dilution of the Donor Plasmid and Resistance Marker Selection

• Transfected HEK293 cells were passaged ten times to dilute out the episomal donor vector.
• After these passages puromycin was added to the media to select for cells with successful knock-in of the RFP-puromycin resistance cassette.
• After 3-4 weeks of selection, >95% of HEK293 cells were expressing RFP.

Figure 3. After transfection, HEK293 cells were passaged ten times to dilute out the episomal vector, then grown in the presence of puromycin for 4 weeks. Top Left) Cells transfected with both pCas-Guide-AAVS1 and pAAVS1-RFP-DNR were healthy after 4 weeks. Top Right) Over 95% of these cells imaged expressed RFP. Bottom Left) Control cells not transfected with the vectors died after puromycin treatment.

Phase #3: Confirmation of Knock-in by Genomic PCR

• To confirm knock-in of RFP in the genomic DNA, a primer pair was designed with Primer 1 targeting the 5’ homology arm upstream of RFP and Primer 2 targeting within the RFP-Puromycin resistance cassette.
• PCR product of 1.1 kb indicates successful knock-in at AAVS1 site; absence of PCR amplification indicates unsuccessful cassette insertion (Figure 4).
• No PCR amplification was seen in the control cells (‘WT cell’) since Primer 2 could not anneal to the genomic DNA.

Figure 4. Genomic PCR was used to confirm the knock-in of RFP. In edited cells, both primer 1 and primer 2 can bind, resulting in a 1.1 kb PCR product. No PCR product is formed in WT cells as primer 2 cannot anneal to the genomic DNA.