CRISPR Stable Knockin Cell Line Generation

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CRISPR Stable Knockin Cell Line Generation
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Cat.No.: C408
Quantity: Basic Service Package
Price: Inquire*
Email: order@abmGood.com
Phone: 604-247-2416 (Local), 1-866-757-2414 (Toll free)
Fax: 604-247-2414
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Orders of this service are subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each order. If you have any questions regarding this, please contact us at technical@abmgood.com. The end user acknowledges that the Materials provided under abm’s Material Transfer Agreement (MTA) does not grant a license for commercial use or imply any ownership rights of any intellectual property rights relating to the Materials.

Knock-in Data

Fluorescence image of HEK293 cell line showing RFP knock-in using CRISPR Cas9 mediated homology directed repair mechanism. Left: RFP knock-in at the AAVS1 (Safe-harbor site) locus. Right: RFP knock-in at a non-AAVS1 site (random insertion).
Service Details
Our Basic Package (C408) Includes Deliverables Lead Time
 A. Target DNA Vector Creation
  • gRNA Design and Construction (2-4 gRNAs)
  • Donor DNA Construction (knock-in or point mutation)
- 5-8 weeks
 B. Cell Culture, Transfection, Optimization - 3-5 weeks
 C. Clonal Selection and Screening
  • KI Confirmation by PCR or Sequencing
- 9-12 weeks
 D. Clonal expansion and cryopreservation Genetically Engineered Cell Line (up to 2 vials 1x106 cells/vial) and Custom Report 3-4 weeks

Add-On Services^ Quantity Cat. No. Price
Cell Line of Your Choice for Gene Editing# 1 Cell Line C141 $800.00
WT Control Cell Line Expressing Cas9 for Comparison
(50% discount for academic customers if ordered with the basic package)
1 Cell Line C142 $1,590.00
Additional Clones 1 Clone C143 $250.00
Additional Vials of Delivered Clones 1 Vial C144 $80.00
Validation Service by Western Blot (Up to 10 Clones)## 1 Service C145 $975.00
Off-Target Analysis by Whole Genome Sequencing Per Clone C146 $4,000.00
Additional rounds of selection and screening by Sanger Sequencing 1 Screening C147 $835.00
HA Tag Validation (Up to 10 Clones)# 1 Service C191 $150.00
STR Profiling of WT and Knock-in cells
(50% discount if ordered with the basic package)
1 Service C287 $150.00
*The cost of the basic service package starts at $15,000 for a single gene knock-in for diploid loci.
**A deposit and MTA is required to initiate all CRISPR Cell Line projects

* 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.
#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.
##100μg of 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 binding sites. 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.
Product Documents
Product References
FAQs
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What is the lead time for a knockout service?
4-5 months for a single gene knock-in.
What should customers provide for this service?
Please supply the cells for this service. For frozen cells, please ship at least 2 vials (at least 10^6 each) on dry ice. An alternative method is to send us two T25 flask of live cells per sample, at 90% confluency. The flasks should be filled with complete medium without any air bubbles and at room temperature. Please ensure that the sterilization procedure is strictly observed. To avoid delays over the weekend, we recommend shipping the cells on a Monday or a Tuesday. Frozen cells are preferred over live cells.

If your cell line already contains an antibiotic resistance marker, please specify. Our vectors have puromycin resistance by default.

If your cells require medium other than DMEM and RPMI, we will also require 1L of the specified medium and any applicable growth factors or supplements to be provided by you for the project. Please submit all components of the complete media (e.g. if growth factors, cytokines etc. are applicable) individually to eliminate potential degradation of components in the media during transit. Kindly include instructions for making the complete media in your shipment. Additionally, please provide at least 5 x coated 6-well plates and 10 x T25 flasks if the cells require specially treated culture vessels. Once you have shipped your cells, please forward us the tracking number for custom clearance. Information on how to ship cell samples to abm can be found on our support page: https://www.abmgood.com/Technical-Support.html

Please place an order first prior to submitting your samples. All samples received must have the order confirmation number indicated. Any samples received without this piece of information will be disposed off immediately upon receipt to ensure that all customer information is held in strict confidence.
Can this service be performed for canine derived cells?
Yes, we can use sgRNA targeting canine genome to make stable KI cell lines. Please provide the specific cell medium for canine cell culture.
What information should I provide to get a quote?
Please complete the Service Questionnaire form under the documents section above and email to quotes@abmgood.com.
Will CRISPR keep cutting the chromosome after the gene is edited?
CRISPR is sensitive to mismatches, so it is unlikely the CRISPR will keep cutting the chromosome after the gene is edited.
How can you detect a mutation if there is no selection (by PCR using primers flanking the modified site or the T7 endonuclease I assay)?
Yes, that is one method used. Other methods include Sanger sequencing, and western blotting if the antibody is provided by the customer.
What are the deliverables?
- Genetically Engineered Cell Line (up to 2 vials 1x10^6 cells/vial)
- Custom Report
Can you offer this service in bacterial cells?
We offer CRISPR knockout and knockin on a custom inquiry basis. Please contact technical@abmgood.com with your project details.
How is KI confirmed?
We perform Sanger sequencing of the locus of interest both to determine the nature of the INDEL in the edited genes to guarantee frameshift silencing, and to confirm that the genome is edited. For even greater certainty, we also offer Next Generation Sequencing confirmation (AI00100), which can detect WT alleles down to 10% of total. Being much more sensitive, NGS screening is ideal for detecting complete editing of multiple copy genes, or polyploid cell lines.