Process of generating knock-out and knock-in cells
PNA Bio offers economic cell line generation (knock-outs and knock-ins) in a timely manner. The process of engineered cell line generation is shown below.
- Design and synthesis of 4~8 gRNA vectors
- Select the most efficient engineered nuclease by mismatch sensitive nuclease assay
- Synthesis of reporter vector for the enrichment of cells with gene editing event
- Optimization of transfection protocol
- Design of donor vector for knock-in project
- Selection of the pool by MACS, FACS, or Hygromycin resistance (option: efficiency validation in a selected pool)
- Single cell cloning
- Screening of the clones using T7E1 assay and/or F-PCR
- Confirmation of homozygous knock out by TA cloning and sequencing
Advantage of our service
- Years of experience using ZFN, TALEN, and CRISPR/Cas9 system
- In house developed software to design gRNA sequences with maximum activity and minimum off-site effect
- Robust assay system to screen and select the most optimal engineered nucleases
- Reporter system to enrich cells with gene editing (3~20 fold improvement over conventional method)
Some of the cell lines that we have generated knock-outs or knock-ins
- Commonly used cells: HEK293, NIH3T3, CHO cells
- Human cancer cells: HeLa, HCT116, SKBR3, Hep3B, HepG2, MCF7, K-562, H-358 cells
- Mouse cells: Pan02, CT26WT cells
- Neuronal cells: Sy5y, PC12 cells
Examples of knock-out clones generated
Example of KO clone generation: sequencing analysis of two KO clones and T7E1 analysis for five off-target sites that contain three mismatches.
We offer the most affordable engineered cell line service in a timely manner.
Knock-out cell line service
- Provide at least two independent KO clones, confirmed for mycoplasma free
- Time line: 4~7 months
Knock-in cell line service
- Provide at least two independent clones, confirmed for mycoplasma free
- Includes donor vector design and synthesis
- Time line: 5~7 months
Confirmation of KO by FACS (example)
- Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence. Mazor Y et al (2017) Sci Rep 7:40098.
- Modeling human epilepsy by TALEN targeting of mouse sodium channel Scn8a. Jones JM & Meisler MH (2014) Genesis 52(2):141-148.
- Small molecules enhance CRISPR genome editing in pluripotent stem cells. Yu C et al (2015) Cell Rep 16(2):142-147.
- Inhibition of non-homologous end joining increases the efficiency of CRISPR/Cas9-mediated precise [TM: inserted] genome editing. Maruyama T et al (2015) Nat Biotech 33(5):538-542.
- ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Yoshimi K et al (2016) Nat Comm 7:10431.
- Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Merkle FT et al (2015) Cell Rep. 11(6):875-883.