KO/KI Cells

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

We have successfully generated knock-out clones up to 5 copies in one round.

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.