Targeted non-viral dna insertions
AssigneeThe Regents of the University of California
InventorsTheodore Lee Roth, Alexander Marson
Life of this patent
Estimating the term…
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.
Claims22
1-28. (canceled)
A modified primary human cell comprising at least one heterologous nucleic acid template non-virally inserted into a target region of the cell's genome, wherein the size of the at least one heterologous nucleic acid template is greater than or equal to 300 nucleotides.
A modified primary human cell comprising at least one heterologous nucleic acid template in a target region of the cell's genome, wherein the size of the nucleic acid template is greater than or equal to 300 nucleotides, and wherein the primary human cell does not comprise a viral vector for introducing the at least one heterologous DNA template into the primary human cell.
A modified primary human cell comprising a ribonucleoprotein (RNP) and a nucleic acid template, wherein the RNP comprises a nuclease domain and a guide RNA, wherein the size of the nucleic acid template is greater than or equal to 300 nucleotides in size, and wherein the 5′ and 3′ ends of the nucleic acid template comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the primary cell, and wherein the primary cell does not comprise a viral vector for delivery of the DNA template.
A method of editing a primary human cell, comprising:
- a. providing a ribonucleoprotein (RNP) and a nucleic acid template, wherein the RNP comprises a nuclease domain and a guide RNA, wherein the size of the nucleic acid template is greater than or equal to 300 bp in size and wherein the 5′ and 3′ ends of the nucleotides template comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the primary cell;
- b. non-virally introducing the RNP and nucleic acid template into the primary cell, wherein the guide RNA specifically hybridizes to a target region of the genome of the primary cell, and wherein the nuclease domain cleaves the target region to create the insertion site in the genome of the primary cell;
- and
- c. editing the primary cell via insertion of the nucleic acid template into the insertion site in the genome of the primary cell.
Dependencies are inferred from the claim text.