Methods for targeted insertion of dna in genes
Methods and compositions for modifying the coding sequence of endogenous genes using rare-cutting endonucleases and transposases. The methods and compositions described herein can be used to modify the coding sequence of endogenous genes.
1. A method of integrating a transgene into an endogenous gene in the genome of a cell, comprising
administering to the cell a first recombinant nucleic acid comprising a transgene comprising in 5′ to 3′ orientation
a first splice acceptor, a first coding sequence, a first terminator, a second terminator reverse complement, a second coding sequence reverse complement, and a second splice acceptor reverse complement; or
a first splice acceptor, a first coding sequence, a bidirectional terminator, a second coding sequence reverse complement, and a second splice acceptor reverse complement;
administering to the cell a second recombinant nucleic acid encoding a CRISPR/Cas9 nuclease having a target site within an intron of the endogenous gene, to thereby provide the CRISPR/Cas9 nuclease in the cell; and
integrating the transgene into the endogenous gene at the CRISPR/Cas9 nuclease target site;
wherein the first coding sequence encodes an amino acid sequence heterologous to the cell and wherein the second coding sequence encodes the same heterologous amino acid sequence; and
wherein following integration the first coding sequence or the second coding sequence is operatively linked to a promoter of the endogenous gene to express a fusion protein comprising the amino acid sequence at its carboxy-terminus.
2. The method of claim 1 , wherein the first recombinant nucleic acid comprises a transgene comprising in 5′ to 3′ orientation a first splice acceptor, a first coding sequence, a first terminator, a second terminator reverse complement, a second coding sequence reverse complement, and a second splice acceptor reverse complement.
3. The method of claim 1 , wherein the first recombinant nucleic acid comprises a transgene comprising in 5′ to 3′ orientation a first splice acceptor, a first coding sequence, a bidirectional terminator, a second coding sequence reverse complement, and a second splice acceptor reverse complement.
4. The method of claim 1 , wherein the transgene is integrated into an intron of the ATXN3 gene.
5. The method of claim 4 , wherein the transgene is integrated into intron 9 of the ATXN3 gene.
6. The method of claim 1 , wherein the transgene is integrated into an intron of the CACNA1A gene.
7. The method of claim 6 , wherein the transgene is integrated into intron 46 of the CACNA1A gene.
8. The method of claim 1 , wherein the transgene is harbored on a viral vector.
9. The method of claim 8 , wherein the viral vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, and a lentivirus vector.
10. The method of claim 8 , wherein the transgene is equal to or less than 4.7 kb in length.