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 which optionally binds to a double-stranded recognition sequence motif. 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 . The engineered chimeric endonuclease of  claim 11 , wherein the first endonuclease variant is an inactive intron-coding homing endonuclease. 
     
     
         13 . The engineered chimeric endonuclease of  claim 12 , wherein the intron-coding endonuclease is selected from the group consisting of “LAGLI-DADG” endonuclease, “His-Cys” Box endonuclease, and “HNH” endonuclease. 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . The engineered chimeric endonuclease of  claim 12 , wherein the nucleic acid binding domain comprises an inactive I-SceI, I-SceII, I-DmoI, I-CreI, I-CeuI, PI-SceI, IPpo, 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 . The engineered chimeric endonuclease of  claim 1 , wherein the cleavage domain comprises at least one catalytic domain of a Type IIS endonuclease. 
     
     
         22 . (canceled) 
     
     
         23 . The engineered nuclease of  claim 21 , wherein the cleavage domain comprises a catalytic domain from a BstF5 I, BtsC I, BsrD I, Bts I, Alw I, Bcc I, BsmA I, Ear I, Mly I, Ple I, Bmr I, Bsa I, BsmB I, Fau I, Mnl I, Sap I, Bbs I, BciV I, Hph I, Mbo II, BfuA I, BspCN I, BspM I, SfaN I, Hga I, BseR I, Bbv I, Eci I, Fok I, BceA I, BsmF I, BtgZ I, BpuE I, Bsg I, Mme I, BseG I, Bse3D I, BseM I, AclW I, Alw26 I, Bst6 I, BstMA I, Eam1104 I, Ksp632 I, Pps I, Sch I, Bfi I, Bso31 I, BspTN I, Eco31 I, Esp3 I, Smu I, Bfu I, Bpi I, BpuA I, BstV2 I, AsuHP I, Acc36 I, Lwe I, Aar I, BseM II, TspDT I, TspGW I, BseX I, BstV1 I, Eco57 I, Eco57M I, Gsu I, or a Bcg I Type IIS endonuclease. 
     
     
         24 . The engineered chimeric endonuclease of  claim 23 , wherein the cleavage domain comprises at least one catalytic domain of a Fok I restriction endonuclease. 
     
     
         25 . The engineered chimeric endonuclease of  claim 24 , wherein the cleavage domain comprises the at least one catalytic domain of a FokI restriction endonuclease associated with at least one portion of a DNA recognition subdomain of the FokI restriction endonuclease. 
     
     
         26 . The engineered chimeric endonuclease of  claim 1 , wherein the nucleic acid binding domain and the nucleic acid cleavage domain are covalently linked without an intervening synthetic peptide linker or with an intervening peptide linker. 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . A recombinant nucleic acid encoding an engineered chimeric endonuclease of  claim 1 .