CRISPR/Cas9 and Targeted Toxins

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.

Histochemical staining of the L1 and L6 spinal cord in
normal rat (A). IB4-conjugated FITC staining of the L6 spinal cord from control (B) and IB4-SAP-treated rats (C) 3 weeks after the treatment. IB4-conjugated FITC staining of the L1 spinal cord from control (D) and IB4-SAP-treated rats (E) 3 weeks after the treatment.
   The lamina II area identified by Nissl’s staining is indicated by dashed lines in A. Note that the staining density of IB4-binding afferent nerve terminals in the lamina II of the L6 spinal cord was depleted after the IB4-SAP treatment.                 Scale bar, 200 μm.