The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

The new frontier of genome engineering with CRISPR-Cas9

DNA is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a non-specific target of cytotoxic agents. Although this is true for traditional chemotherapeutics, other agents that were discovered more recently have shown enhanced efficacy. Furthermore, a new generation of agents that target DNA-associated processes are anticipated to be far more specific and effective. How have these agents evolved, and what are their molecular targets?

https://faculty.missouri.edu/~gatesk/DNAasaTarget.pdf

DNA and its associated processes as targets for cancer therapy

Chromosomal translocations in the human acute leukemias rearrange the regulatory and coding regions of a variety of transcription factor genes. The resultant protein products can interfere with regulatory cascades that control the growth, differentiation, and survival of normal blood cell precursors. Support for this interpretation comes from the results of gene manipulation studies in mice, as well as the sequence homology of oncogenic transcription factors with proteins known to regulate embryonic development in primitive organisms, including the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Many of these genetic alterations have important prognostic implications that can guide the selection of therapy. The insights gained from studies of translocation-generated oncogenes and their protein products should hasten the development of highly specific, and hence less toxic, forms of leukemia therapy.

http://www.cs.duke.edu/brd/Teaching/Previous/Bio/Readings/aml.pdf

Oncogenic Transcription Factors in the Human Acute Leukemias

How a cell responds to stress is a central problem in cardiovascular biology. Diverse physiological stresses (eg, heat, hemodynamics, mutant proteins, and oxidative injury) produce multiple changes in a cell that ultimately affect protein structures and function. Cells from different phyla initiate a cascade of events that engage essential proteins, the molecular chaperones, in decisions to repair or degrade damaged proteins as a defense strategy to ensure survival. Accumulative evidence indicates that molecular chaperones such as the heat shock family of stress proteins (HSPs) actively participate in an array of cellular processes, including cytoprotection. The versatility of the ubiquitous HSP family is further enhanced by stress-inducible regulatory networks, both at the transcriptional and posttranscriptional levels. In the present review, we discuss the regulation and function of HSP chaperones and their clinical significance in conditions such as cardiac hypertrophy, vascular wall injury, cardiac surgery, ischemic preconditioning, aging, and, conceivably, mutations in genes encoding contractile proteins and ion channels.

Stress (Heat Shock) Proteins: Molecular Chaperones in Cardiovascular Biology and Disease

Recognition of the DNA minor groove by pyrrole-imidazole polyamides.

Small molecules that target specific DNA sequences have the potential to control gene expression. Ligands designed for therapeutic application must bind any predetermined DNA sequence with high affinity and permeate living cells. Synthetic polyamides containing N-methylimidazole and N-methylpyrrole amino acids have an affinity and specificity for DNA comparable to naturally occurring DNA-binding proteins. We report here that an eight-ring polyamide targeted to a specific region of the transcription factor TFIIIA binding site interferes with 5S RNA gene expression in Xenopus kidney cells. Our results indicate that pyrrole-imida-zole polyamides are cell-permeable and can inhibit the transcription of specific genes.

Regulation of gene expression by small molecules

SMALL molecules that specifically bind with high affinity to any predetermined DNA sequence in the human genome would be useful tools in molecular biology and potentially in human medicine Simple rules have been developed to control rationally the sequence specificity of minor-groove-binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids Two eight-ring pyrrole–imidazole polyamides differing in sequence by a single amino acid bind specifically to respective six-base-pair target sites which differ in sequence by a single base pair Binding is observed at subnanomolar concentrations of ligand The replacement of a single nitrogen atom with a C-H regulates affinity and specificity by two orders of magnitude The broad range of sequences that can be specifically targeted with pyrrole–imidazole polyamides, coupled with an efficient solid-phase synthesis methodology, identify a powerful class of small molecules for sequence-specific recognition of double-helical DNA

Recognition of DNA by designed ligands at subnanomolar concentrations

Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole–imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located within RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

https://www.pnas.org/content/pnas/95/22/12890.full.pdf

Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands.

The sequence-specific recognition of the minor groove of DNA by pyrrole−imidazole polyamides has been extended to 9−13 base pairs (bp). Four polyamides, ImPyPy-Py-PyPyPy-Dp, ImPyPy-G-PyPyPy-Dp, ImPyPy-β-PyPyPy-Dp, and ImPyPy-γ-PyPyPy-Dp (Im = N-methylimidazole, Py = N-methylpyrrole, Dp = N,N-dimethylaminopropylamide, G = glycine, β = β-alanine, and γ = γ-aminobutyric acid), were synthesized and characterized with respect to their DNA-binding affinities and specificities at sequences of composition 5‘-(A,T)G(A,T)_5C(A,T)-3‘ (9 bp) and 5‘-(A,T)_5G(A,T)C(A,T)_5-3‘ (13 bp). In both sequence contexts, the β-alanine-linked compound ImPyPy-β-PyPyPy-Dp has the highest binding affinity of the four polyamides, binding the 9 bp site 5‘-TGTTAAACA-3‘ (K_a = 8 × 10^8 M^(-1)) and the 13 bp site 5‘-AAAAAGACAAAAA-3‘ (K_a = 5 × 10^9 M^(-1)) with affinities higher than the formally N-methylpyrrole-linked polyamide ImPyPy-Py-PyPyPy-Dp by factors of ∼8 and ∼85, respectively (10 mM Tris·HCl, 10 mM KCl, 10 mM MgCl_2, and 5 mM CaCl_2, pH 7.0). The binding data for ImPyPy-γ-PyPyPy-Dp, which has been shown previously to bind DNA in a “hairpin” conformation, indicates that γ-aminobutyric acid does not effectively link polyamide subunits in an extended conformation. These results expand the binding site size targetable with pyrrole−imidazole polyamides and provide structural elements that will facilitate the design of new polyamides targeted to other DNA sequences.

Extension of sequence-specific recognition in the minor groove of DNA by pyrrole-imidazole polyamides to 9-13 base pairs

Cell-permeable small molecules that bind predetermined DNA
sequences with affinities and specificities comparable to those
of natural DNA-binding proteins have the potential to regulate
the expression of specific genes. Recently, an eight-ring hairpin
pyrrole-imidazole (Py-Im) polyamide which binds six base pairs
of DNA was shown to inhibit transcription of a specific gene in
cell culture. Polyamides recognizing longer DNA sequences
should provide more specific biological activity. To specify a
single site within the 3 billion base pair human genome, ligands
which specifically recognize 15-16 base pairs are necessary.
For this reason, recognition of 16 base pairs represents a milestone
in the development of chemical approaches to DNA recognition.
We examine here the affinity and specificity of a Py-Im
polyamide dimer which targets 16 contiguous base pairs in the
minor groove of DNA.

John W. Trauger

Papers

Regulation of gene expression by small molecules

Recognition of DNA by designed ligands at subnanomolar concentrations

Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands.

Extension of sequence-specific recognition in the minor groove of DNA by pyrrole-imidazole polyamides to 9-13 base pairs

Recognition of 16 base pairs in the minor groove of dna by a pyrrole-imidazole polyamide dimer