Research Publications

Research – bioRxiv Pre-print – Enhancing Cas9 Activity in Heterochromatin

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Enhancing Cas9 Activity in Heterochromatin
Daer R, Barrett C, Haynes KA. (2017) bioRxiv. https://www.biorxiv.org/content/early/2017/12/04/228601

CRISPR is a powerful and popular tool for editing DNA in living cells. Scientists are becoming more interested in using CRISPR to correct mistakes in DNA that lead to diseases, to artificially generate mutations to research the origins of diseases, and for other important applications. However, CRISPR originated in bacteria and has probably not evolved to function very well in genomes that are packed in configurations (open and closed chromatin) as complex as those found in human cells. In a recent report (Daer et al. 2017), we demonstrated that CRISPR activity was inhibited at a DNA sequence that became artificially condensed into closed chromatin. Our new study shows that targeted re-opening of closed chromatin leads to enhanced CRISPR activity in the same region. The epigenetic drug we tested (UNC1999) was not sufficient to generate a transcriptionally active or CRISPR-accessible state. In contrast, strong direct activation with a DNA-binding p65 protein did enhance CRISPR accessibility. Importantly, we learned that a recovery period (following treatment with p65) is needed to generate the CRISPR-accessible state.

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Research – bioRxiv Pre-print – Activation of tumor suppressor genes in breast cancer cells by a synthetic chromatin effector

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Activation of tumor suppressor genes in breast cancer cells by a synthetic chromatin effector
Olney KC, Nyer DB, Wilson Sayres MA, Haynes KA. (2017) bioRxiv. http://www.biorxiv.org/content/early/2017/09/07/186056

Certain types of breast cancer can be difficult to treat because breast cancer cells in different patients are not completely identical. Here, we measured the expression levels of genes in drug responsive and non-responsive (triple-negative) lab-grown breast cancer cells. In agreement with findings from other research groups, we observed that certain groups of genes are commonly or differentially expressed. Importantly, a large group of genes is silenced in breast cancer cells compared to less cancerous cells. In cancers, certain overactive proteins silence genes by inducing tight chromatin packing. We used a synthetic fusion protein called PcTF to bind and disrupt cancer-associated gene silencing. Dozens of genes, including fifteen different anti-cancer genes became activated in all of the cancer cell types, including the triple-negative cells. PcTF has the potential to act as a powerful therapeutic protein (biologic) that activates multiple anti-cancer genes at once.

Research – bioRxiv Pre-print – Tandem histone binding domains enhance the activity of a synthetic chromatin effector

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Tandem histone-binding domains enhance the activity of a synthetic chromatin effector
Tekel SJ, Vargas DA, Song L, LaBaer J, Haynes KA. (2017) bioRxiv. http://biorxiv.org/content/early/2017/06/03/145730

Here, we report the behavior of a re-engineered PcTF,  a gene-regulating fusion protein that is designed to activate genes that have been suppressed by chromatin condensation in cancer cells. We added an extra histone-binding domain to create Pc2TF and observed 2- to 4-fold enhancement of target binding and target gene activation. The new design was inspired by natural proteins that also have double-motifs that contribute to target affinity. The specific combination of motifs in Pc2TF does not exist in nature. By using design rules inferred from pre-existing motif patterns, we have improved the performance a novel synthetic chromatin effector. This improved activity advances PcTF towards clinical translation for anti-cancer therapy.

Related resources:

  1. In Vitro Development of Synthetic Chromatin Proteins That Function in Live Cells. FASEB. Abstract. http://www.fasebj.org/content/31/1_Supplement/922.8.short

Research – ACS Synthetic Biology – The impact of chromatin dynamics on Cas9-mediated genome editing in human cells

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BioRxiv_Daer2016The impact of chromatin dynamics on Cas9-mediated genome editing in human cells
Daer RM, Cutts JP, Brafman DA, Haynes KA (2016) ACS Synthetic Biology. doi: 10.1021/acssynbio.5b00299

We used a chromatin switch system to compare the efficiency of human gene editing (via CRISPR/Cas9) before and after DNA had become packaged with nuclear proteins. The DNA-nuclear protein complex (called chromatin) ‘turns the dials’ of gene expression. Here, we discovered that this dialing mechanism can also disrupt artificial genome editing. We also found that readjusting chromatin could restore gene editing, which has implications for improving CRISPR for use in stem cell genomes, where key genes are often tightly packaged in chromatin.

Related resources:

Research – Nature – Regulation of cancer epigenomes with a histone-binding synthetic transcription factor

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Figure6_finalRegulation of cancer epigenomes with a histone-binding synthetic transcription factor.
Nyer DB, Daer R, Vargas D, Hom C, Haynes KA. (2017) Nature Genomic Medicine. http://rdcu.be/oqv7

This work expands our 2011 report in many important ways. We studied the behavior of a synthetic chromatin protein that we designed called PcTF in bone, blood, and brain cancer-derived cells. We expected to see PcTF bind to methylated histones, but instead saw strong signals closer to histone-free gene promoters. However, PcTF activity still required the methyl-histone binding domain to interact with its targets. It appears that PcTF bridges methylated histone signals with the transcription complex. We also discovered that PcTF activates a key tumor suppressor, CASZ1 as well as other silenced genes in all three cancer cell types. This new information has advanced our understanding of how a potentially therapeutic histone-binding protein behaves in cancer cells.

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Research – Biotechnology & Bioengineering – The histone deacetylase inhibitor Entinostat enhances polymer-mediated transgene expression in cancer cell lines

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Abstract_figureThe histone deacetylase inhibitor entinostat enhances polymer-mediated transgene expression in cancer cell lines
Elmer JJ, Christensen MD, Barua S, Lehrman J, Haynes KA, Rege K. (2015) Biotechnol Bioeng. PMID: 26614912

Collaborators from the Haynes Lab (ASU, SBHSE), Rege Lab (ASU, SEMTE), and Elmer Lab (Villanova) published our discovery of how drugs that modify epigenetic mechanisms improve the expression of synthetic genes that are delivered into cultured human cells. Dr. Elmer is the first author. Dr. Haynes and Matt Christensen (Rege Lab) used quantitative PCR to discover that treatment with a low molecular weight compound called Entinostat led to increased uptake of synthetic DNA into the nuclei of cells.

 

Research – ACS Syn Bio – A sensitive switch for visualizing natural gene silencing in single cells

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ACSsynbio2011A sensitive switch for visualizing natural gene silencing in single cells
Haynes KA, Ceroni F, Flicker D, Younger A, Silver PA. (2012) ACS Synth. Biol. 1: 99–106. PMID: 22530199

We designed a synthetic gene switch that expressed cyan fluorescent protein in the presence of microRNAs, which are biomarkers for cell development and disease. The switch was designed to be sensitive to small, hard-to-detect microRNAs in live cells. Here, we demonstrated that the switch responded to natural as well as artificial proof-of-concept microRNA signals.