Targeted non-viral dna insertions
AssigneeThe Regents of the University of California
InventorsTheodore Lee Roth, Alexander Marson
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Abstract
Provided herein are methods and compositions for editing the genome of a cell. In some embodiments, a nucleotide sequence of at least 200 nucleotides in length is inserted into a target region in the genome of a cell.
Claims20
A primary immune cell comprising at least one DNA template non-virally inserted into a target region of the genome of the cell, wherein the size of the DNA template is greater than or equal to about 200 base pairs (bp).
A primary immune cell comprising at least one DNA template inserted into a target region of the genome of the primary immune cell, wherein the size of the DNA template is greater than or equal to 200 base pairs, and wherein the primary immune cell does not comprise a viral vector for introducing the DNA template into the primary immune cell.
A viable, virus-free, primary cell comprising a ribonucleoprotein complex (RNP)-DNA template complex, wherein the RNP comprises a nuclease domain and a guide RNA, wherein the size of the DNA template is greater than or equal to 200 nucleotides in size, and wherein the 5′ and 3′ ends of the DNA template comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the primary cell.
A method of editing a primary immune cell, comprising:
- a. providing a ribonucleoprotein complex (RNP)-DNA template complex, wherein the RNP comprises a nuclease domain and a guide RNA, wherein the size of the DNA template is greater than or equal to 200 nucleotides in size, and wherein the 5′ and 3′ ends of the DNA template comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the primary immune cell;
- b. non-virally introducing the RNP-DNA template complex into the primary immune cell, wherein the guide RNA specifically hybridizes to a target region of the genome of the primary immune cell, and wherein the nuclease domain cleaves the target region to create the insertion site in the genome of the primary immune cell;
- and
- c. editing the primary immune cell via insertion of the DNA template into the insertion site in the genome of the primary immune cell.
Dependencies are inferred from the claim text.