An engineered Fok I cleavage half-domain, wherein the engineered cleavage half-domain comprises one or more mutations as follows: (a) the wild-type residue at position 479 is replaced with a Gln (Q) or Thr (T) residue (I479Q or I479T); (b) the wild-type residue at position 418 is replaced with a Glu (E) or Asp (D) residue (S418E or S418D); (c) the wild-type residue at position 446 is replaced with an Asp (D) residue (S446D); (d) the wild-type residue at position 448 is replaced with an Ala (A) residue (K448A); (e) the wild type residue at position 481 is replaced with an Ala (A), Glu (E), Cys (C), or Ser (S) residue (Q481A, Q481E, Q481C or Q481S); (f) the wild-residue at position 416 is replaced with a Glu (E), Asp (D), His (H) or Asn (N) residue (R416E, R416D, R416H or R416N); (g) the wild-type residue at position 422 is replaced with a His (H) residue (R422H); (h) the wild-type residue at position 424 is replaced with a Phe (F) residue (L424F); (i) the wild-type residue at position 472 is replaced with an Asp (D) residue (S472D); (j) the wild-type residue at position 476 is replaced with a Glu (E) residue or a Gly (G) residue (N476E or N476G); (k) the wild-type residue at position 478 is replaced with an Asp (D) residue (P478D); (l) the wild-type residue at position 480 is replaced with an Asp (D) residue (G480D); (m) the wild-type residue at position 525 is replaced with an Ala (A), Cys (C), Glu (E), Ile (I), Ser (S), Thr (T) or Val (V) residue (K525A, K525C, K525E, K525I, K525S, K525T or K525V); and/or (n) the wild-type residue at position 542 is replaced with an Asp (D) residue (N542D), wherein the amino acid residues are numbered relative to full length Fok I wild-type cleavage domain, as shown in SEQ ID NO:1. (a) the wild-type residue at position 479 is replaced with a Gln (Q) or Thr (T) residue (I479Q or I479T); (b) the wild-type residue at position 418 is replaced with a Glu (E) or Asp (D) residue (S418E or S418D); (c) the wild-type residue at position 446 is replaced with an Asp (D) residue (S446D); (d) the wild-type residue at position 448 is replaced with an Ala (A) residue (K448A); (e) the wild type residue at position 481 is replaced with an Ala (A), Glu (E), Cys (C), or Ser (S) residue (Q481A, Q481E, Q481C or Q481S); (f) the wild-residue at position 416 is replaced with a Glu (E), Asp (D), His (H) or Asn (N) residue (R416E, R416D, R416H or R416N); (g) the wild-type residue at position 422 is replaced with a His (H) residue (R422H); (h) the wild-type residue at position 424 is replaced with a Phe (F) residue (L424F); (i) the wild-type residue at position 472 is replaced with an Asp (D) residue (S472D); (j) the wild-type residue at position 476 is replaced with a Glu (E) residue or a Gly (G) residue (N476E or N476G); (k) the wild-type residue at position 478 is replaced with an Asp (D) residue (P478D); (l) the wild-type residue at position 480 is replaced with an Asp (D) residue (G480D); (m) the wild-type residue at position 525 is replaced with an Ala (A), Cys (C), Glu (E), Ile (I), Ser (S), Thr (T) or Val (V) residue (K525A, K525C, K525E, K525I, K525S, K525T or K525V); and/or (n) the wild-type residue at position 542 is replaced with an Asp (D) residue (N542D), wherein the amino acid residues are numbered relative to full length Fok I wild-type cleavage domain, as shown in SEQ ID NO:1. <EOS>