A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality.

BACKGROUND: Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. RESULTS: Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. CONCLUSION: This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection.

Imidazo[5,1-f][1,2,4]triazin-2-amines as novel inhibitors of polo-like kinase 1.

The synthesis and biological activities of imidazo[5,1-f][1,2,4]triazin-2-amines (imidazotriazines) as novel polo-like kinase 1 inhibitors are reported.

Mechanism of enantioselective Ti-catalyzed Strecker reaction: peptide-based metal complexes as bifunctional catalysts.

Kinetic, structural, and stereochemical data regarding the mechanism of Ti-catalyzed addition of cyanide to imines in the presence of Schiff base peptide ligands are disclosed. The reaction is first order in the Ti-ligand complex; kinetic studies reveal DeltaS(dagger) = -45.6 +/- 4.1 cal K(-1) mol(-1), indicating a highly organized transition structure for the turnover-limiting step of the catalytic cycle. A mechanistic model consistent with the kinetic and stereochemical data is presented, where the Ti center is coordinated to the Schiff base unit of the ligand and the AA2 moiety of the peptidic segment of the chiral ligand associates and delivers HNC to the activated bound substrate. Thus, these studies illustrate that these non-C2-symmetric catalysts likely operate in a bifunctional fashion.

Combinatorial catalyst discovery.

There have been recent attempts to use the principles of combinatorial chemistry and high-throughput screening strategies for catalyst identification. With the technology available that allows the synthesis of large libraries, scientists of varied backgrounds have implemented screening efforts to identify active and selective catalysts. Within this context, several techniques have come to light in the past year: infrared thermography is used to identify optimal catalysts by monitoring the change in temperature for exothermic reactions; fluorescence and colored-dye assays, a familiar tool to biologists, is being applied to the identification of catalysts that exhibit the highest activity. Whereas none of these screening methods provide a general solution to the problem of screening large combinatorial libraries (there is likely to be no general solution), each advance represents an important intellectual and technological step forward.