TCRα homing endonuclease variants
The present disclosure provides improved genome editing compositions and methods for editing a TCRα gene.
1. A polypeptide comprising an I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human T cell receptor alpha (TCRα) gene, wherein the I-OnuI HE variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8.
2. The polypeptide of claim 1 , wherein the I-OnuI HE variant comprises the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8.
3. The polypeptide of claim 1 , wherein the polypeptide binds and cleaves said TCRα gene at the polynucleotide sequence set forth in SEQ ID NO: 17.
4. The polypeptide of claim 1 , further comprising a TALE DNA binding domain.
5. The polypeptide of claim 4 , wherein the TALE DNA binding domain comprises 11.5 TALE repeat units, and wherein the TALE DNA binding domain binds the polynucleotide sequence set forth in SEQ ID NO: 19.
6. The polypeptide of claim 4 , wherein the TALE DNA binding domain comprises 10.5 TALE repeat units, and wherein the TALE DNA binding domain binds the polynucleotide sequence set forth in SEQ ID NO: 18.
7. The polypeptide of claim 4 , wherein the polypeptide binds and cleaves said TCRα gene at the polynucleotide sequence set forth in SEQ ID NO: 20.
8. The polypeptide of claim 1 or claim 4 , further comprising:
(a) a peptide linker or a viral self-cleaving 2A peptide; and
(b) a 3′-5′ exonuclease.
9. The polypeptide of claim 8 , wherein the 3′-5′ exonuclease is Trex2.
10. A polypeptide, comprising an I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human T cell receptor alpha (TCRα) gene and further comprising a TALE DNA binding domain, wherein the polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 12.
11. The polypeptide of claim 4 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12.
12. A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an I-OnuI homing endonuclease (HE) variant that cleaves a target site in the human T cell receptor alpha (TCRα) gene, wherein the I-OnuI HE variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8.
13. A vector comprising the polynucleotide of claim 12 .
14. A cell comprising the polynucleotide of claim 12 .
15. The polynucleotide of claim 12 , wherein the nucleotide sequence encoding the polypeptide further comprises a nucleotide sequence encoding a TALE DNA binding domain.