Resources AND Seminars

 Enabling Tools for Genome Engineering


Our laboratory has been striving to improve the delivery, quality, and efficiency of CRISPR/Cas9 editing systems guided by  experimental approaches. To this end, we have repurposed CRISPR/Cas9 technology for in vivo genetic screens (PMID 24298059), assessed parameters that influence Cas9 on-target versus off-target cleavage efficiency (PMID 25275497), identified small molecule modulators that favor DNA repair by homologous recombination over non-homologous end joining (PMID 26307031), and identified PAM multiplicity within target sites as being detrimental to Cas9 editing efficiency (PMID 26644285). Lentivirus and Retroviral vectors optimized for co-expression of Cas9 and sgRNAs and harboring fluorescent protein reporters are readily available and distributed to colleagues (Fig 1).

To extend the utility of Cas9 in vivo, we we have designed an inducible transgenic mouse which will express Cas9 across all tissues analyzed upon administration of Doxycycline. This allows for the design of sgRNA-delivery vectors of a dramatically-reduced size and facilitates a wide-variety of in vivo genome engineering experiments.

Chemical Biology Resources

RNA helicases have been explored as drug targets, but mainly in the context of antiviral strategies. One strategy to target these has been to use ring-expanded 'fat' nucleosides and nucleotides or benzimidazoles and benzotriazoles. However the specificity of this approach remains unproven since nucleoside analogues are capable of forming tight complexes with nucleic acid substrates and can also target other NTP binding proteins. Our laboratory has amassed a unique collection of natural products, from which we identified threee potent and highly selective eIF4A inhibitors following an HTS screening campaign (PMID 16030146, 16532013, 18551192). These compounds (hippuristanol, rocaglates, and pateamine A) have very different mechanisms of action. Two of these, pateamine A and silvestrol, behave as chemical inducers of dimerization (CIDs) – forcing an engagement between RNA and eIF4A - resulting in sequestration of eIF4A from the eIF4F complex. On the other hand, hippuristanol inhibits eIF4A from binding to RNA. In Myc-driven lymphomas displaying elevated mTOR signaling, silvestrol and hippuristanol re-sensitize tumors to the cytotoxic effects of doxorubicin. Importantly, B-cells are more sensitive to silvestrol when derived from chronic lymphocytic leukemia patients than from healthy individuals, suggesting that leukemic or faster-growing cells are more sensitive to translation inhibition. Our research program has shown that blocking translation initiation by targeting eIF4A sensitizes cells to killing by cytotoxic agents (e.g.-doxorubicin). This body of work illustrates that selective pharmacological inhibition of RNA helicases is very much possible. We have collaborated with many labs worldwide with these compounds - either in providing access to  these unique reagents or expertise on their use/effects in vitro and in vivo (see below for a map tthe distribution of our compounds). 

Unique GEMMs that Mimic Pharmacological Target Suppression

We have generated novel GEMMs in which shRNAs can be conditionally expressed to knockdown the expression of their targets in virtually any tissue. These powerful GEMMs can be used to mimic pharmacological suppression of their targets to  discern any resulting phenotypes (therefore, potential drug side-effects) as well as provide systems-wide insight into any perturbed expression pathways. Mice suppressing expression of eIF4E or the RNA helicase, DHX9, have been generated and are currently under study. These unique reagents are available to interested investigators.

Small Molecule Inhibitors of Translation

Among the unique Know-how present in my laboratory garnered by my Trainees is a comprehensive understanding of small molecules that target protein synthesis. Below we provide an up-to-date table of translation initiation inihibitors and their known mechanism of action (MOI). Compounds in red are those identified by our laboratory as being translation inhibitors.

Eukaryotic Initiation Inhibitors

1. Cap Analogs (eg - m7GDP, m7GTP, m7GpppG, 7-benzyl-GMP, 4Ei-1)

     MOI: Inhibits eIF4E-mediated recruitment of 43S pre-initiation complexes to mRNAs (PMID 22495651).

2. 4E1RCat, 4E2RCat, 4E3RCat

     MOI: Interferes with eIF4E:eIF4G Interaction (PMID 21191102)

3. 4EGI-1

     MOI: Interferes with eIF4E:eIF4G binding and stabilizes unphosphorylated 4E-BP1:eIF4E binding (PMID 26170285)

4. Hippuristanol

     MOI: Prevents eIF4AI and eIF4AII from binding to RNA (PMID 16532013)

5. Rocaglates

     MOI: Interface inhibitor that stimulates eIF4A:RNA binding - depleting it from the eIF4F complex (PMID 18551192, 19401772)

6. Pateamine A

     MOI: Interface inhibitor that stimulates eIF4A:RNA binding - depleting it from the eIF4F complex (PMID 16030146, 16337595)

7. 4E ASO4  

     MOI: Targets the 3’ untranslated region of eIF4E and triggers RNAse H-mediated RNA destruction.

 5’-TGTCATATTCCTGGATCCTT-3’; MOE-modified bases are in red

8. SBI-756

     MOI: Targets eIF4GI and disrupts the eIF4F complex (PMID 26603897).

9. NSC 119889, NSC 119893

     MOI: Inhibits ternary complex formation by targeting eIF2 (PMID 14769948, 16928960).

10. MDMP (2-(4-methyl-2, 6-dinitroanilino)-N-methylpropionamide)

     MOI: Blocks 40S/60S subunit joining (PMID 4198116).

11. NSC 218351, NSC 92218

     MOI: Decreases the fidelity of start site recognition (PMID 21220547).

12. Edeine

     MOI: Interferes with AUG codon recognition by scanning ribosomes (PMID 681367)