The Combinational Use of CRISPR/Cas9 and Targeted Toxin Technology Enables Efficient Isolation of Bi-Allelic Knockout Non-Human Mammalian Clones. Watanabe S, Sakurai T, Nakamura S, Miyoshi K, & Sato M. (2018). Int J Mol Sci, 19 (4).
Objective: Most genome editing systems employ transient treatment with selective drugs such as puromycin to obtain the desired genome-edited cells, which often allows some untransfected cells to survive and decreases the efficiency of generating genome-edited cells. The authors developed a novel targeted toxin-based drug-free selection system for the enrichment of genome-edited cells.
Summary: Results indicate that a combination of the CRISPR/Cas9 system and targeted toxin technology using IB4-SAP allows efficient enrichment of genome-edited clones, particularly bi-allelic KO clones.
Dose: Cells were trypsinized 3 days after transfection and approximately 80% were incubated for 30 min at 37°C in a solution (25 mcL) containing 0.5–1.0 mcg IB4-SAP (Cat. #IT-10).
IB4-SAP (Cat. #IT-10)
A tool for eliminating cells that express α-D-galactopyranoside residues in cells; targeted via recombinant Isolectin B4 (IB4), eliminated via saporin.
Isolectin B4 (IB4) is one of a family of five alpha-D-galactose-binding lectins from Griffonia (Bandeiraea) simplicifolia. Recombinant IB4* was expressed in E. coli and purified using affinity chromatography. In one important application rIB4-SAP specifically eliminates the IB4-positive c-fiber nociceptor neurons, while sparing the peptidergic neurons. Upon binding the alpha-D-galactopyranoside residues expressed on the cell surface, rIB4-SAP becomes internalized and saporin inhibits protein synthesis, resulting in elimination of the neurons.