Rationally designed meganucleases with sequence specificity and altered DNA binding affinity

摘要

A method for producing a recombinant meganuclease that has specificity for a semisite of altered recognition sequence that differs by at least one base pair from a semisite within a recognition sequence of the I-Crel meganuclease selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, said method comprising: A. designing a polypeptide sequence having a similarity of at least 85% with residues 2-153 of the I-Crel meganuclease of SEQ ID NO: 1, said design comprising modifying the sequence of residues 2-153 of the I-Crel meganuclease of SEQ ID NO: 1, where (1) the specificity in the position -1: (a) to a T in a coding strand by a modification selected from the group consisting of C70, L70, Y75, Q75, H75, H139, Q46 and H46; (b) to an A in a coding strand by a modification selected from the group consisting of Y75, L75, C75, Y139, C46 and A46; (c) to a G in a coding strand by a modification selected from the group consisting of E70, E75, E46 and D46; (d) to a C in a coding strand by a modification selected from the group consisting of H75, R75, H46, K46 and R46; or (e) to any base in a coding strand by a modification of G46; and / or (2) the specificity in a position -2 has been altered: (a) to an A in a coding strand 81 by a modification selected from the group consisting of V44, I44, L44 and N44; (b) to a C in a coding strand by a modification of E70; (c) to an A or a T in a coding strand by a modification comprising C44; and / or (3) the specificity in a position -3 has been altered: (a) to an A in a coding strand by a modification of C24; (b) to a C in a coding strand by a modification selected from the group consisting of F68, K24 and R24; (c) to a T in a coding strand by a modification selected from the group consisting of M68, C68, L68 and F68; (d) to a C or a T in a coding strand by a modification comprising Y68; and / or (4) the specificity in a position -4 has been altered: (a) to a C in a coding strand by a modification selected from the group consisting of E77 and K26; (b) to a G in a coding strand by a modification selected from the group consisting of E26 and R77; (c) to a C or a T in a coding strand by a modification comprising S77; or (d) to any base in a coding strand by means of a modification comprising S26, and / or (5) the specificity in a position -5 has been altered: (a) to a C in a coding strand by a modification comprising E42; (b) to a G in a coding strand by a modification comprising R42; (c) to an A or a G in a coding strand by a modification selected from the group consisting of C28 and Q42; or (d) to any base in a coding strand by a modification selected from the group consisting of M66 and K66; and / or (6) the specificity in a -6 position has been altered: (a) to a T in a coding strand by a modification selected from the group consisting of 60 C40, I40, V40, C79, I79, V79 and Q28; (b) to a C in a coding strand by a modification selected from the group consisting of E40 and R28; or (c) to a G in a coding strand by a modification comprising R40; and / or (7) the specificity in a position -7 has been altered: (a) to a C in a coding strand by a modification selected from the group consisting of E38, K30 and R30; (b) to a G in a coding strand by a modification selected from the group consisting of K38, R38 and E30; (c) to a T in a coding strand by a modification selected from the group consisting of I38 and L38; or (d) to an A or a G in a coding strand by a modification comprising C38; or (e) to any base in a coding strand by a modification selected from the group consisting of H38, N38 and Q30; and / or (8) the specificity in a position -8 has been altered: (a) to a T in a coding strand by a modification selected from the group consisting of L33, V33, I33, F33 and C33; (b) to a C in a coding strand by a modification selected from the group consisting of E33 and D33; (c) to a G in a coding strand by a modification consisting of K33; or (d) to an A or a C in a coding strand by a modification comprising R32; or (e) to an A or a G in a coding strand by a modification comprising R33; and / or (9) the specificity in a position -9 has been altered: (a) to a C in a coding strand by a modification comprising E32; (b) to a G in a coding strand by a modification selected from the group consisting of R32 and K32; (c) to a T in a coding strand by a modification selected from the group consisting of L32, V32, A32 and C32; (d) to a C or a T in a coding strand by a modification selected from the group consisting of D32 and I32; or (e) to any base in a coding strand by a modification selected from the group consisting of S32, N32, H32, Q32 and T32, wherein said polypeptide sequence does not comprise residues 2-153 of the I-Crel meganuclease SEQ ID NO: 1 only with the following modifications that alter the specificity: N75, A68 / N70 / N75, D44 / K70 / N75, R44 / AG8 / T70 / N75, A44 / L70 / N75, A44 / D68 / K70 / N75, A44 / G68 / N75, K44 / T68 / G70 / N75, D44 / N70 / N75, A44 / T68 / S70 / N75, R44 / N70 / N75, A44 / S68 / N75, A26, C26, C26 / R66, R66, C33, H33, T68 / K70 / N75, K70 / N75, N70 / N75, T70 / N75, A70 / N75, T44 / N75, A68 / K70 / N75, N68 / K70 / N75, G70 / N75, SG8 / K70 / N75, N68 / N75, H70 / N75, S70 / N75, G68 / K70 / N75, Q70 / N75, Q68 / K70 / N75, H68 / K70 / N75, A32, A140, F33, A30, A44, A38, A44 / A140, K32, R32, N33, R33, A68, A33, G33, I33, K33, L33, P33, Q33, S33, T33, A28, A70, D33, E33, M33, V33 and W33; and B. producing a recombinant meganuclease comprising said polypeptide sequence.