Engineered nucleases and their uses for nucleic acid assembly

Abstract

Aspects of the invention provide engineered endonucleases that are characterized by both a long recognition sequence and specific cleavage outside of the recognition site. Engineered endonucleases of the invention are useful for manipulating long pieces of DNA.

1 . An engineered chimeric endonuclease comprising:
 a nucleic acid binding domain of a first endonuclease linked to a nucleic acid cleavage domain of a second endonuclease,   wherein the nucleic acid binding domain binds a recognition sequence motif recognized by the first endonuclease and is free of an active catalytic domain of the first endonuclease, and   wherein the nucleic acid cleavage domain cleaves at a unique cleavage position outside of the recognition motif.   
     
     
         2 . The engineered chimeric endonuclease of  claim 1 , wherein the nucleic acid binding domain is a DNA binding domain. 
     
     
         3 . The engineered chimeric endonuclease of  claim 1 , wherein the nucleic acid binding domain binds to a double-stranded recognition sequence motif. 
     
     
         4 . The engineered chimeric endonuclease of  claim 1 , wherein the nucleic acid binding domain binds specifically to a unique double-stranded recognition sequence motif. 
     
     
         5 . The engineered chimeric endonuclease of  claim 1 , wherein the nucleic acid binding domain binds selectively to several related recognition sequence motifs. 
     
     
         6 . The engineered chimeric endonuclease of  claim 1 , wherein the nucleic acid binding domain binds with nanomolar affinity to a target nucleic acid comprising the recognition sequence motif. 
     
     
         7 . The engineered chimeric endonuclease of  claim 1 , wherein the recognition sequence motif has a length of 8 to 10, 10 to 20, 20 to 40, 40-100, or 100-200 nucleotides. 
     
     
         8 . The engineered chimeric endonuclease of  claim 1 , further comprising an inactive mutant catalytic domain of the first endonuclease. 
     
     
         9 . The engineered chimeric endonuclease of  claim 8 , wherein the nucleic acid binding domain of the first endonuclease comprises the inactive mutant catalytic domain. 
     
     
         10 . The engineered chimeric endonuclease of  claim 1 , wherein the nucleic acid binding domain comprises a meganuclease nucleic acid binding domain. 
     
     
         11 . The engineered chimeric endonuclease of  claim 8 , wherein the first endonuclease is a meganuclease variant, and the inactive mutant catalytic domain comprises a catalytic site having one or more amino acid substitutions that inactivate the catalytic endonuclease activity. 
     
     
         12 .- 16 . (canceled) 
     
     
         17 . The engineered chimeric endonuclease of  claim 8 , wherein the nucleic acid binding domain comprises an inactive I-SceI, I-SceII, I-DmoI, I-CreI, I-CeuI, PI-SceI, I-Ppo, I-TevI, I-TevII, I-TevIII, I-CeuI, or PspI binding domain. 
     
     
         18 . The engineered chimeric endonuclease of  claim 17 , wherein the inactive variant I-Sce endonuclease comprises an N at position 44 and an A at position 145. 
     
     
         19 . The engineered chimeric endonuclease of  claim 17 , wherein the inactive variant I-Sce endonuclease comprises an A at position 44 and an A at position 145. 
     
     
         20 . The engineered chimeric endonuclease of  claim 17 , wherein the inactive variant I-Cre endonuclease comprises an N at position 20 and an A at position 47. 
     
     
         21 .- 32 . (canceled) 
     
     
         33 . A recombinant nucleic acid encoding an engineered chimeric endonuclease of  claim 1 . 
     
     
         34 . A recombinant host cell comprising the recombinant nucleic acid of  claim 33