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Principal Investigator: Omar O Abudayyeh
Organization: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Fiscal Year: 2021
Award: $568,123
Funding agency: National Institute of Biomedical Imaging and Bioengineering
Project Summary
Despite extraordinary advances in genome engineering, tools for precise and efficient gene correction across all
cell types and desired edits remain lacking. Current programmable DNA cleavage tools, such as CRISPR-Cas9,
rely on cellular DNA repair mechanisms, which are inefficient and do not function in post-mitotic cells. Thus,
genome editing still needs efficient, robust tools that can make a variety of specific DNA sequence alterations.
These tools could have broad applications across both basic biological discovery, allowing for new modalities of
screening, and therapeutics, including engineered cell therapies. The proposed work will address these needs
by combining computational discovery, biochemical characterization, and enzyme engineering to develop
integrase-based tools for programmable, multiplexed insertion of large genes in diverse cell types
independent of DNA repair. The discovery, characterization, and engineering of these new integrase proteins
will both build upon our deep history of CRISPR enzyme discovery, as well as draw from new, high-throughput
approaches to mine biological diversity. Complementary to the discovery of these new enzymes, we will combine
Cas9-based genome editing with integrase engineering to develop programmable, multiplexed genome
integration systems that do not depend on DNA repair mechanisms, allowing integration of large sequences in
any cell type. We will explore delivery mechanisms, including viruses, electroporation, and novel lipid
nanoparticle formulations to edit T cells and neurons. We will engineer aspects of the integrases, including
protein engineering and site mutagenesis, to boost activity of the system and screen many insertion sites to
develop design rules for the technology. Moreover, through studying orthogonal integrases sites we can develop
multiplexed versions of the insertion tool to edit up to three sites in a given cell with superior efficiency over other
tools. We will apply these multiplexed integrases to develop a new screening system, where tagging of multiple
genes can be used for determining protein interaction partners in high throughput. Our new integrase systems
will also be applied to the development of multiple-edited T-cells for improved immuno-oncology therapies. The
multiple technologies resulting from these discoveries and engineering efforts will overcome the limitations of
existing genome and epigenome engineering approaches and serve as a valuable resource for broader
biomedical research. Programmable gene integration with CRISPR-recruited integrases will allow for more
advanced genome engineering applications to be pursued in cells and in vivo, accelerating the pace of
biomedical research, enabling greater exploration of basic biological processes and disease mechanisms, and
promoting novel therapeutic developments.
Terms: <Address><Assay><Bacteriophages><Basal Transcription Factor><Basal transcription factor genes><Bio-Informatics><Bioassay><Biochemical><Biodiversity><Bioinformatics><Biologic Assays><Biological><Biological Assay><Biological Diversity><Biological Function><Biological Process><Biology><Biomedical Research><CRISPR><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 system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Line><Cell Therapy><Cell model><CellLine><Cells><Cellular model><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Complex><Confocal Microscopy><DNA><DNA Alteration><DNA Damage Repair><DNA Repair><DNA Sequence Alteration><DNA mutation><Deoxyribonucleic Acid><Development><Disease><Disease model><Disorder><Electroporation><Elements><Engineering><Enzyme Gene><Enzymes><Formulation><Future><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Alteration><Genetic Change><Genetic Diversity><Genetic Variation><Genetic defect><Genetic mutation><Genetics-Mutagenesis><Genome><Genome engineering><Guide RNA><History><Immune><Immunes><Immunooncology><Integrase><Interphase Cell><Location><Mammalian Cell><Mediating><Methodology><Mining><Mitotic><Modality><Modification><Mutagenesis><Mutagenesis Molecular Biology><Mutation><Nerve Cells><Nerve Unit><Neural Cell><Neurocyte><Neurons><Non-Polyadenylated RNA><Non-dividing Cell><Nondividing Cell><Pathway interactions><Phages><Position><Positioning Attribute><Protein Engineering><Proteins><RNA><RNA Gene Products><Recording of previous events><Regulatory Element><Research Resources><Resources><Resting Cell><Ribonucleic Acid><Sequence Alteration><Site><Strains Cell Lines><Study models><System><Systems Integration><T-Cells><T-Lymphocyte><Technology><Testing><Therapeutic><Therapeutic Uses><Transcription Factor Proto-Oncogene><Transcription factor genes><Unscheduled DNA Synthesis><Virus><Work><bacterial virus><base><biological research><cell engineering><cell mediated therapies><cell type><cell-based therapeutic><cell-based therapy><cellular engineering><cellular therapy><computational pipelines><cultured cell line><cytokine><design><designing><developmental><disorder model><electroporative delivery><epigenome><gRNA><gene correction><gene electrotransfer><gene-corrected><genetic protein engineering><genome editing><genome mutation><genomic alteration><genomic editing><human disease><immune-oncology><immuno oncology><immunology oncology><improved><in vitro Model><in vivo><indel><innovate><innovation><innovative><insertion-deletion><insertion-deletion mutation><insertion/deletion><insertion/deletion mutation><insight><lipid nanoparticle><neuronal><new drug treatments><new drugs><new technology><new therapeutics><new therapy><next generation therapeutics><novel><novel drug treatments><novel drugs><novel technologies><novel therapeutics><novel therapy><oncoimmunology><pathway><programs><protein design><protein protein interaction><recruit><repair><repaired><response><screening><stress granule><therapeutic agent development><therapeutic development><thymus derived lymphocyte><tool><transcription factor>