A Small-Molecule Mask for Traceless Protein Delivery

NIH Pandemic-Era Grants

Pandemic Era Grants

2019

↗ Live URL (may be dead)

Document text

Principal Investigator: Lucas William Erickson
Organization: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Fiscal Year: 2019
Award: $52,868
Funding agency: National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT
The CRISPR/Cas9 system has quickly emerged as the most robust gene editing tool yet discovered. Many
early papers employing this system have utilized a plasmid that encodes the Cas9 protein and the guide RNA
(sgRNA). These plasmids are typically delivered in viral shells that can often be too small for the entire
plasmid, requiring multiple segments to be transported separately. Another drawback of plasmid incorporation
is the continual expression of the protein and RNA, which often leads to undesired off-target effects. A
promising alternative is the delivery of the Cas9·sgRNA complex directly. A variety of methods have been
developed for the delivery of this complex, including lipid nanoparticles and cell-penetrating peptides, but all
methods so far result in inefficient gene editing or are inapplicable in vivo (or both). Recent work in our lab has
demonstrated that the unique chemistry of the benzoxaborole functional group allows it to deliver proteins
efficiently and directly into the cytosol. Combining this unique chemistry with the reactivity of a diazo motif will
allow us to develop small molecules that can mask Cas9 carboxylates via an esterification reaction. A series of
these delivery vehicles will be synthesized and reacted with the Cas9·sgRNA complex to evaluate their effect
on complex stability and protein–RNA binding. Initial experiments will focus on knocking out the GFP gene in
GFP-producing HEK cells. Once the optimal delivery vehicle and conditions have been determined, that
vehicle will be employed as a tool in future studies of breast cancer metastasis. Collagen prolyl 4-hydroxylase
has been demonstrated to be significant in the metastasis process, and we anticipate that knock-out of this
gene via a CRISPR/Cas9 system would be an effective approach. Finally, our delivery strategy could provide a
straightforward method for delivering not only Cas9·sgRNA complexes, but also a wide variety of proteins.

Terms: <Adoption><Animals><Assay><Binding><Bioassay><Biologic Assays><Biological Assay><Breast Metastasis><Breast cancer metastasis><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Coat><Cell Line><CellLine><Cells><Chemicals><Chemistry><Cleaved cell><Collagen><Complex><Cytosol><DNA><DNA delivery><Deoxyribonucleic Acid><Development><Drug Precursors><Ester Hydrolase><Esterification><Fluorescence><Future><Gene Transcription><Genes><Genetic Transcription><Glycocalyx><Goals><Guide RNA><Half-Life><Human><In Vitro><Investigators><Isoelectric Point><Knock-out><Knockout><Label><Length><Masks><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Metastasis><Metastasize><Metastatic Lesion><Metastatic Mass><Metastatic Neoplasm><Metastatic Tumor><Methods><Modern Man><Molecular Interaction><Neoplasm Metastasis><Non-Polyadenylated RNA><Paper><Peptides><Peptidyl Prolyl Hydroxylase><Plasmids><Pro-Drugs><Process><Procollagen Prolyl 4-Hydroxylase><Procollagen-Proline Dioxygenase><Prodrugs><Proline Hydroxylase><Proline,2-Oxoglutarate 4-Dioxygenase><Prolyl 4-Hydroxylase><Prolyl Hydroxylase><Proteins><Protocollagen Prolyl Hydroxylase><RNA><RNA Expression><RNA Gene Products><RNA-Binding Proteins><Reaction><Reagent><Research Personnel><Researchers><Ribonucleic Acid><Secondary Neoplasm><Secondary Tumor><Series><Strains Cell Lines><System><Techniques><Technology><Therapeutic><Transcription><Transfection><Viral><Work><base><cancer metastasis><carboxylate><cleaved><cultured cell line><deliver DNA><design><designing><developmental><esterase><experiment><experimental group><experimental research><experimental study><functional group><gRNA><genome editing><genomic editing><gold nano particle><gold nanoparticle><guanidinium><in vivo><lipid nanoparticle><nano gold><nanoGold><plasmid DNA><protein expression><side effect><small molecule><success><tool><tumor cell metastasis><uptake>