Meganuclease variants cleaving a dna target sequence from the mouse rosa26 locus and uses thereof

Abstract

An I-CreI variant, wherein one of the two I-CreI monomers has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated respectively from positions 26 to 40 and 44 to 77 of I-CreI, said variant being able to cleave a DNA target sequence from the mouse ROSA26 locus. Use of said variant and derived products for the engineering of transgenic mice and recombinant mouse cell lines expressing an heterologous protein of interest.

1 - 43 . (canceled) 
     
     
         44 . A method of cleaving a DNA target sequence from a mouse ROSA26 locus comprising contacting said DNA target sequence with an I-CreI variant to thereby cleave said DNA target sequence
 wherein said I-CreI variant comprises a first monomer and a second monomer which are associated to form an active form,   wherein said I-CreI variant comprises at least two substitutions in at least one of the monomers,   wherein at least one substitution is of a residue in the range of positions 26 to 40 of I-CreI and at least one substitution is of a residue in the range of positions 44 to 77 of I-CreI and   wherein said DNA target sequence is at least one sequence selected from the group consisting of SEQ ID NO: 5 to 14 and 16 to 30.   
     
     
         45 . The method of  claim 44 , wherein said at least one substitution of a residue in the range of 26 to 40 of I-CreI is at least one substitution of a residue selected from the group consisting of positions 26, 28, 30, 32, 33, 38 and 40. 
     
     
         46 . The method of  claim 44 , wherein said at least one substitution of a residue in the range of 44 to 77 of I-CreI is at least one substitution of a residue selected from the group consisting of positions 44, 68, 70, 75 and 77. 
     
     
         47 . The method of  claim 44 , wherein said substitutions comprise replacing the wild-type amino acids with an amino acid selected from the group consisting of A, D, E, G, H, K, N, P, Q, R, S, T, Y, C, W, L and V. 
     
     
         48 . The method of  claim 44 , wherein said I-CreI variant further comprises at least one substitution selected from the group consisting of: G19S, I24V, S79G, V105A, K107R, V151A, D153G and K158E. 
     
     
         49 . The method of  claim 44 , wherein said I-CreI variant further comprises at least one substitution at positions 137 to 143 of I-CreI. 
     
     
         50 . The method of  claim 44 , which comprises substitution of the aspartic acid in position 75 of I-CreI. 
     
     
         51 . The method of  claim 50 , wherein position 75 of I-CreI is substituted with an asparagine residue. 
     
     
         52 . The method of  claim 44 , wherein said variant is a homodimer. 
     
     
         53 . The method of  claim 44 , wherein said variant is a heterodimer, resulting from the association of a first and a second monomer having different mutations in positions 26 to 40 and 44 to 77 of I-CreI. 
     
     
         54 . The method of  claim 53 , wherein the first and the second monomer of said I-CreI variant, respectively, comprise at least one pair of substitutions selected from the group consisting of:
 N30H, Y33S, Q44E, R68c, R70S, D75N and N30D, Y33R, Q38T, Q44K, R68E, R70S, I77R,   S32N, Y33G, Q44K, R70E, D75N and S32T, Q38W, Q44K, R68E, R70S, I77R,   Y33R, Q38N, S40Q, Q44N, R70S, D75R, I77D and N30H, Y33S, Q44A, R70S, D75Q, I77E,   K28S, Q38R, S40K, Q44D, R68Y, R70S, D75S, I77R and Y33C, Q38A, R68A, R70K, D75N,   Y33C, Q44T, R70S, D75Y and S32D, Q38C, Q44D, R68Y, R70S, D75S, I77R,   S32T, Y33C, R68T, R70N, D75N and S32T, Q38W, Q44K, R70E, D75N,   R70S, D75R, I77Y and Y33R, Q38A, S40Q, Q44A, R70S, D75N,   K28S, Q38R, S40K, Q44T, R68N, R70N, D75N and Y33H, Q38S, Q44K, R68Y, R70S, D75Q, I77N,   K28A, Y33S, Q38R, S40K, Q44N, R68Y, R70S, D75R, I77V and S32T, Y33C, Q44A, R70S, D75N,   S32D, Y33H, Q44K, R68E, R70S, I77R and S32D, Y33H, Q44D, R68N, R70S, D75N,   N30R, S32D, R68S, R70K, D75N and D75N,   K28R, Y33A, Q38Y, S40Q, R68Y, R70S, D75R, I77Q and Y33R, Q38A, S40Q, R70S, I77K,   K28R, N30D, D75E, I77R and S32D, Q38C, Q44A, R70S, D75R, I77Y,   Y33R, Q38A, S40Q, R70S, D75N and R70S, D75Y, I77R,   Y33R, Q38A, S40Q, Q44N, R68Y, R70S, D75R, I77V and N30R, S32D, Q44T, R68H, R70H, D75N,   Y33P, S40Q, Q44K, R68Y, R70S, D75Q, I77N and S32A, Y33C, R68Y, R70S, D75R, I77Q,   K28A, Y33S, Q38R, S40K, R68N, R70S, D75N, I77R and S32N, Y33G, Q44A, R68A, R70K, D75N,   N30H, Y33S, Q44Y, R70S, I77V and Y33R, S40Q, Q44A, R70S, D75N,   S32D, Y33H, R68T, R70N, D75N and N30R, S32D, Q44T, R68N, R70N, D75N,   S32R, Y33D, Q44A, R70S, D75N and Y33S, Q38R, S40H, R68H, R70H, D75N,   Y33P, S40Q, Q44K, R68Y, R70S, D75Q, I77N and K28R, Y33A, Q38Y, S40Q, Q44T, R68H, R70H, D75N,   K28Q, Q38R, S40K, Q44A, R70S, D75E, I77R and N30R, S32D, Q44K, R68Y, R70S, D75Q, I77N,   Y33T, Q38A, R68H, R70H, D75N and N30H, Y33S, R70S, I77K,   Y33T, S40T, R68A, R70K, D75N and K28R, Y33A, Q38Y, S40Q, R70S, and   S32D, Y33H, Q44N, R70S, D75R, I77D and S32D, Q38C, R70S, I77K.   
     
     
         55 . The method of  claim 53 , wherein the first monomer is selected from the group consisting of SEQ ID NO: 82 to 106 and the second monomer is selected from the group consisting of SEQ ID NO: 107 to 116, 4, and 117 to 130. 
     
     
         56 . The method of  claim 53 , wherein the target sequence is SEQ ID NO: 15. 
     
     
         57 . The method of  claim 56 , wherein the first monomer has substitutions selected from the group consisting of: I24V, Q44Y, R70S and D75N; I24V, Q44Y, R68Y, R70S, D75Y and I77R; I24V, Q44Y, R70S, D75N and I77V; I24V, Q44Y, R68N, R70S and D75R; I24V, Q44Y, R68S, R70S and D75R; I24V, Q44Y, R70S and D75Q; I24V, Q44Y, R68Y, R70S, D75R and I77V; I24V, Q44Y, R70S, D75Y and I77T, and the second monomer has substitutions selected from the group consisting of: K28E, Y33R, Q38R, S40R, Q44A, R68H, R70Q and D75N; K28E, Y33R, Q38R, S40R, Q44A, R70N and D75N; K28E, Y33R, Q38R, S40K, Q44A, R68H, R70Q and D75N; K28E, Y33R, Q38R, S40K, Q44V, R70A and D75N; K28E, Y33R, Q38R, S40K, Q44A, R70G and D75N. 
     
     
         58 . The method of  claim 57 , wherein the first monomer is selected from the group consisting of SEQ ID NO: 39, 43, 45, 49, 50, 51, 53 and 54, and the second monomer is selected from the group consisting of SEQ ID NO: 60, 61, 63, 65 and 66. 
     
     
         59 . The method of  claim 58 , wherein at least one of the two I-CreI monomers has at least 95% sequence identity with, for the first monomer, SEQ ID NO: 39, 43, 45, 49, 50, 51, 53 or 54, and, for the second monomer, SEQ ID NO: 60, 61, 63, 65 or 66. 
     
     
         60 . The method of  claim 56 , wherein the first and the second monomer, respectively, comprise at least the following substitutions: I24V, Q44Y, R70S, D75Y, I77T and K28E, Y33R, Q38R, S40R, Q44A, R68S, R70Q, D75N. 
     
     
         61 . The method of  claim 60 , wherein the first and the second monomers are SEQ ID NO:54 and SEQ ID NO: 62, respectively. 
     
     
         62 . The method of  claim 61 , wherein at least one of the two I-CreI monomers has at least 95% sequence identity with, for the first monomer, SEQ ID NO: 54, and, for the second monomer, SEQ ID NO: 62. 
     
     
         63 . The method of  claim 56 , wherein the first monomer has substitutions selected from the group consisting of: I24V, Q44Y, R68N, R70S and D75R; I24V, Q44Y, R68S, R70S and D75R; I24V, Q44Y, R70S and D75Q; I24V, Q44Y, R68Y, R70S, D75R and I77V; I24V, Q44Y, R70S, D75Y and I77T, and the second monomer has substitutions selected from the group consisting of: K28E, Y33R, Q38R, S40K, Q44A, R70S, D75N and K28E, Y33R, Q38R, S40K, Q44A, R68T, R70N, D75N. 
     
     
         64 . The method of  claim 63 , wherein the first monomer is selected from the group consisting of the sequences SEQ ID NO: 49, 50, 51, 53 and 54, and the second monomer is selected from the group consisting of the sequences SEQ ID NO: 64 and 67. 
     
     
         65 . The method of  claim 64 , wherein at least one of the two I-CreI monomers has at least 95% sequence identity with, for the first monomer, SEQ ID NO: 49, 50, 51, 53 or 54, and, for the second monomer, SEQ ID NO: 64 or 67. 
     
     
         66 . The method of  claim 56 , wherein the first monomer is selected from the group consisting of the sequences SEQ ID NO: 72 and 73 and the second monomer is selected from the group consisting of the sequences SEQ ID NO: 74 to 77. 
     
     
         67 . The method of  claim 66 , wherein at least one of the two I-CreI monomers has at least 95% sequence identity with, for the first monomer, SEQ ID NO: 72 or 73, and, for the second monomer, SEQ ID NO: 74, 75, 76, or 77. 
     
     
         68 . The method of  claim 53 , wherein the first monomer further comprises the D137R mutation and the second monomer further comprises the R51D mutation. 
     
     
         69 . The method of  claim 53 , wherein the first monomer further comprises the E8R or E8K and E61R mutations and the second monomer further comprises the K7E and K96E mutations. 
     
     
         70 . The method of  claim 44 , wherein said variant is a single-chain chimeric meganuclease comprising two I-CreI monomers. 
     
     
         71 . The method of  claim 70 , wherein said chimeric meganuclease comprises a first monomer and a second monomer wherein each monomer has the same substitutions. 
     
     
         72 . The method of  claim 70 , wherein said chimeric meganuclease comprises a first monomer and a second monomer wherein each monomer has at least one different substitution in positions 26 to 40 and 44 to 77 of I-CreI. 
     
     
         73 . The method of  claim 44 , wherein said I-CreI variant is made from the starting scaffold of SEQ ID NO: 1. 
     
     
         74 . The method of  claim 44 , wherein said I-CreI variant is made from the starting scaffold of SEQ ID NO: 133. 
     
     
         75 . The method of  claim 44 , wherein said I-CreI variant is made from the starting scaffold of SEQ ID NO: 4. 
     
     
         76 . The method of  claim 44  wherein said contacting is in a cell. 
     
     
         77 . The method of  claim 44  wherein said I-CreI variant is expressed in a cell from a polynucleotide encoding said I-CreI variant.