Rationally designed meganucleases with sequence specificity and altered DNA binding affinity

摘要

A method for cleaving a double-stranded DNA target recognition sequence into a eukaryotic cell comprising: introducing into said eukaryotic cell a nucleic acid encoding a meganuclease; or a meganuclease protein; wherein said meganuclease cleaves said double stranded DNA target recognition sequence; and wherein said meganuclease is a recombinant meganuclease comprising: a polypeptide having at least 85% sequence similarity with residues 2-153 of the I-CreI meganuclease of SEQ ID NO: 1; and which has specificity for a recognition sequence semisite that differs by at least one base pair from a semisite in an I-CreI meganuclease recognition sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO : 3, SEQ ID NO: 4 and SEQ ID NO: 5, where the specificity at position -1: (i) has been altered to an A by a modification selected from the group consisting of D75C, D74L, D75Y, K139Y, T46A and T46C; (ii) to a G through a modification selected from the group consisting of T46E, T46D and T75E; or (iii) to a T by a modification selected from the group consisting of D75H, D75Q, D75Y, K139H, T46H and T46Q; (iv) to any base in a strand felt by a T46G modification. with the proviso that the method is not a method for the treatment of the human body or an animal by therapy; with the proviso that the method is not a method to modify the genetic identity of the germ line of human beings and with the additional caveat that the cell is not a human embryonic stem cell or a human blastocyst cell.