Coupling endonucleases with end-processing enzymes drives high efficiency gene disruption
The present disclosure relates to the co-expression of an endonuclease with an end-processing enzyme for the purpose of enhanced processing of the polynucleotide ends generated by endonuclease cleavage.
1. A method of increasing mutagenesis of a nucleotide sequence at a selected nucleic acid site in a cell comprising: selecting a sequence for targeted mutagenesis in said cell; selecting an endonuclease that cleaves at said selected nucleic acid site; and coupling the activity of said endonuclease and the activity of one or more end-processing enzymes within said cell so as to increase mutagenesis at said selected nucleic acid site.
2. The method of claim 1 wherein the endonuclease is engineered to recognize the nucleotide sequence.
3. The method of claim 1 wherein the end-processing enzyme exhibits 5-3′ exonuclease, 5-3′ alkaline exonuclease, 3-5′exonuclease, 5′ flap endonuclease, helicase, or template-independent DNA polymerases activity.
4. The method of claim 1 wherein the nucleotide sequence encodes CCR-5 or a fragment thereof.
5. The method of claim 1 wherein the end-processing enzyme is Trex2 or a biologically active fragment thereof.
6. The method of claim 1 wherein a fusion protein having endonuclease and end-processing activity is provided.
7. The method of claim 6 wherein the fusion protein comprises a linker domain.
8. The method of claim 1 wherein one or more proteins having endonuclease activity and one or more proteins having end-processing activity are provided.
9. The method of claim 8 wherein the one or more proteins having endonuclease activity and the one or more proteins having end-processing activity are encoded by one or more expression vectors.
10. The method of claim 4 wherein the one or more proteins having endonuclease activity and the one or more proteins having end-processing activity are encoded by a single expression vector comprising a T2A sequence.
11. The method of claim 6 wherein at least one protein comprises a zinc finger domain.
12. The method of claim 8 wherein one or more polypeptides having end-processing activity is an exonuclease or a biologically active fragment thereof.
13. The method of claim 8 wherein one or more polypeptides having end-processing activity is Trex2 exonuclease or a biologically active fragment thereof.
14. The method of claim 8 wherein one or more polypeptides comprise a zinc finger domain.