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AssigneePresident and Fellows of Harvard College
InventorsGeorge M. Church, Oscar Castanon Velasco, Cory J. Smith
Estimating the term…
The present disclosure provides highly multiplexed base editing methods and compositions that minimize the induction of DNA damage sensors in eukaryotic cells and maintain cell viability. The disclosed base editing methods improve the survival of eukaryotic cells after large-scale genome editing. These methods are based upon the discovery that use of a dead Cas9 base editor and optimal cell conditions during and after base editing enhances cells' tolerance to and survival following thousands of edits to the genome. Optimal cell conditions after base editing include the use of a combination of small molecule factors and/or inhibitors. These methods are facilitated by the design and use of tens to hundreds to thousands of gRNAs for guiding the base editor to the target sequences. The disclosed methods are capable of inducing between ten and 300,000 edits to the genome of a eukaryotic cell. Further disclosed are pharmaceutical compositions and compositions of eukaryotic cells comprising fusion proteins and a plurality of unique gRNAs, and a combination of small molecule factors and inhibitors. Also disclosed are kits for the generation of the fusion protein-gRNA complexes described herein.
A method of base editing comprising:
A method of base editing comprising:
A method of base editing comprising:
A method of base editing comprising:
A method of base editing comprising:
A method of base editing comprising:
A composition of eukaryotic cells comprising a plurality of fusion proteins, wherein each of the fusion proteins of the plurality comprises (i) a nuclease inactive Cas9 (dCas9) domain and (ii) a deaminase domain, and a guide RNA (gRNA) bound to the dCas9 domain, wherein at least five of the fusion proteins of the plurality are each bound to a unique gRNA comprising a different guide sequence of at least 10 contiguous nucleotides that is complementary to a target sequence in the genomic DNA of the cells.
A pharmaceutical composition comprising a plurality of fusion proteins, wherein each of the fusion proteins of the plurality comprises (i) a nuclease inactive Cas9 (dCas9) domain and (ii) a deaminase domain, and a guide RNA (gRNA) bound to the dCas9 domain, wherein at least five of the fusion proteins of the plurality are each bound to a unique gRNA comprising a different guide sequence of at least 10 contiguous nucleotides that is complementary to a target sequence in the genomic DNA of a eukaryotic cell, and a pharmaceutically acceptable excipient.
A pharmaceutical composition comprising a plurality of fusion proteins, wherein each of the fusion proteins of the plurality comprises (i) a TAL effector domain, (ii) a deaminase domain, and (iii) a cofactor protein associated with the TAL effector domain, wherein at least five of the fusion proteins of the plurality are each bound to a unique cofactor protein that binds to a target sequence in the genomic DNA of a eukaryotic cell, and a pharmaceutically acceptable excipient.
A kit comprising a nucleic acid construct, comprising
A kit comprising a nucleic acid construct, comprising
A kit comprising a nucleic acid construct, comprising
Dependencies are inferred from the claim text.