[go: up one dir, main page]

WO2006097784A1 - Variants de meganuclease i-crei presentant une specificite modifiee, leur procede de preparation, et leurs utilisations - Google Patents

Variants de meganuclease i-crei presentant une specificite modifiee, leur procede de preparation, et leurs utilisations Download PDF

Info

Publication number
WO2006097784A1
WO2006097784A1 PCT/IB2005/000981 IB2005000981W WO2006097784A1 WO 2006097784 A1 WO2006097784 A1 WO 2006097784A1 IB 2005000981 W IB2005000981 W IB 2005000981W WO 2006097784 A1 WO2006097784 A1 WO 2006097784A1
Authority
WO
WIPO (PCT)
Prior art keywords
crel
site
meganuclease variant
positions
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2005/000981
Other languages
English (en)
Inventor
Frédéric PAQUES
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cellectis SA
Original Assignee
Cellectis SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cellectis SA filed Critical Cellectis SA
Priority to PCT/IB2005/000981 priority Critical patent/WO2006097784A1/fr
Priority to ES06744673T priority patent/ES2347684T3/es
Priority to US11/908,934 priority patent/US20110158974A1/en
Priority to CA2600033A priority patent/CA2600033C/fr
Priority to PCT/IB2006/001271 priority patent/WO2006097854A1/fr
Priority to AT06744673T priority patent/ATE466933T1/de
Priority to EP10004689A priority patent/EP2327771A1/fr
Priority to EP10004688A priority patent/EP2327773A1/fr
Priority to DK06744673.2T priority patent/DK1863909T3/da
Priority to AU2006224248A priority patent/AU2006224248B2/en
Priority to PCT/IB2006/001203 priority patent/WO2006097853A1/fr
Priority to DE602006014107T priority patent/DE602006014107D1/de
Priority to EP10004717A priority patent/EP2327772A1/fr
Priority to JP2008501447A priority patent/JP2008535484A/ja
Priority to US11/908,798 priority patent/US7897372B2/en
Priority to CN200680012709.7A priority patent/CN101198694B/zh
Priority to EP06744673.2A priority patent/EP1863909B2/fr
Priority to EP10004687A priority patent/EP2325307A1/fr
Publication of WO2006097784A1 publication Critical patent/WO2006097784A1/fr
Anticipated expiration legal-status Critical
Priority to US12/859,905 priority patent/US20110072527A1/en
Priority to US13/422,902 priority patent/US8715992B2/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention relates to a method of preparing ⁇ -CreJ meganuclease variants, with a modified specificity, i.e. able to cleave at least one homing site that is not cleaved by the wild-type ⁇ -Cre ⁇ .
  • the invention relates also to the l-Crel meganuclease variants obtainable by said method and to their applications either for cleaving new DNA target or for genetic engineering and genome engineering for non-therapeutic purposes.
  • the invention also relates to nucleic acids encoding said variants, to expression cassettes comprising said nucleic acids, to vectors comprising said expression cassettes, to cells or organisms, plants or animals except humans, transformed by said vectors.
  • Meganucleases are sequence specific endonucleases recognizing large (>12bp; usually 14-40 bp) DNA cleavage sites (Thierry and Dujon, 1992). In the wild, meganucleases are essentially represented by homing endonucleases, generally encoded by mobile genetic elements such as inteins and class I introns (Belfort and Roberts, 1997; Chevalier and Stoddard, 2001). Homing refers to the mobilization of these elements, which relies on DNA double-strand break (DSB) repair, initiated by the endonuclease activity of the meganuclease.
  • DSB DNA double-strand break
  • Homing endonucleases fall into 4 separated families on the basis of pretty well conserved amino acids motifs [for review, see Chevalier and Stoddard (Nucleic Acids Research, 2001 , 29, 3757-3774)].
  • One of them is the dodecapeptide family (dodecamer, DOD, D1-D2, LAGLIDADG, P1-P2). This is the largest family of proteins clustered by their most general conserved sequence motif: one or two copies (vast majority) of a twelve-residue sequence: the dodecapeptide.
  • Homing endonucleases with one dodecapetide (D) are around 20 IcDa in molecular mass and act as homodimers.
  • DD Those with two copies
  • DD range from 25 kDa (230 amino acids) to 50 kDa (HO, 545 amino acids) with 70 to 150 residues between each motif and act as monomer.
  • Cleavage is inside the recognition site, leaving 4 nt staggered cut with 3'OH overhangs.
  • Enzymes that contain a single copy of the LAGLIDADG motif, such as l-Ceul and ⁇ -Crel act as homodimers and recognize a nearly palindromic homing site.
  • ⁇ -CreI pdb accession code Ig9y
  • ⁇ -Crel comprises 163 amino acids (pdb accession code Ig9y); said endonuclease cuts as a dimer.
  • the LAGLIDADG motif corresponds to residues 13 to 21 ; on either side of the LAGLIDADG ⁇ -helices, a four ⁇ -sheet (positions 21-29; 37- 48; 66-70 and 73-78) provides a DNA binding interface that drives the interaction of the protein with the half-site of the target DNA sequence.
  • the dimerization interface involves the two LAGLIDADG helix as well as other residues.
  • the homing site recognized and cleaved by l-Crel is 22-24 bp in length and is a degenerate palindrome (see figure 2 of Jurica MS et al, 1998 and SEQ ID NO:65).
  • said l-Crel homing site is a semi-palindromic 22 bp sequence, with 7 of 1 1 bp identical in each half-site (Seligman LM et al., NAR, 2002, 30, 3870-3879).
  • the endonuclease-DNA interface has also been described (see figure
  • - homing site sequence must have at least 20 bp to achieve a maximal binding affinity of 0.2 nM;
  • R51 and K98 are located in the enzyme active site and are candidates to act as Lewis acid or to activate a proton donor in the cleavage reaction; mutations in each of these residues have been observed to sharply reduce l-Crel endonu- cleolytic activity (R51 G, K98Q);
  • meganuclease-induced DSB stimulates homologous recombination up to 10 000-fold
  • meganucleases are today the best way to improve the efficiency of gene targeting in mammalian cells (Choulika et al., 1995; Cohen-Tannoudji et al., 1998; Donoho et al, 1998; Elliott et al, 1998; Rouet et al, 1994), and to bring it to workable efficiencies in organisms such as plants (Puchta et al, 1993; Puchta et al, 1996) and insects (Rong and Golic, 2000; Rong and Golic, 2001 ; Rong et al, 2002).
  • Meganucleases have been used to induce various kinds of homologous recombination events, such as direct repeat recombination in mammalian cells (Liang et al, 1998), plants (Siebert and Puchta, 2002), insects (Rong et al, 2002), and bacteria (Posfai et al, 1999), or interchromosomal recombination (Moynahan and Jasin, 1997; Puchta, 1999; Richardson et al, 1998).
  • Homing endonucleases have also been used as scaffolds to make novel endonucleases, either by fusion of different protein domains (Chevalier et al.,
  • LAGLIDADG homing endonuclease (Chevalier B. et al., 2004) or for providing I-
  • mutants The kinetic behavior and DNA binding properties of these mutants were assessed. More specifically, the following nine mutants were prepared: D20N, D20L, D20A; Q47N, Q47A, Q47M, Q47E; K98K and K98A. Mutations at residue D20 demonstrate greater heterogeneity in their effect on DNA affinity (wild-type 1-CreJ target site) than those mutations at Q47.
  • - Seligman LM et al., 2002 describe mutations altering the cleavage specificity of ⁇ -Cre ⁇ . More specifically, they have studied the role of the nine amino acids of 1-Crel predicted to directly contact the DNA target (Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70).
  • mutants which have been designed and constructed have each of said nine amino acids and a tenth (Tl 40) predicted to participate in a water-mediated interaction, converted to alanines.
  • - Q26A, R68A and Y33A are inactive, - K28A and R70A are inactive and non-toxic.
  • Seligman et al. have also studied the interaction between I-Crel position 33 and homing site bases 2 and 21 or between 1-Crel position 33 and homing site bases 1 and 22.
  • the 1-Crel mutants analysed when displaying an increased affinity for a mutant homing site also displayed an activity for the wild- type homing site (Table 1 and figure 3 of Seligman) even though said activity is decreased.
  • - homing site mutants altered at the positions 2/21, 3/20, 7/16, 8/15 and 9/14 are resistant to cleavage by wild-type ⁇ -CreI in vivo; however, in vitro assay using E. coli appears to be more sensitive than the in vivo test and allows the detection of homing sites of wild-type ⁇ -Crel more effectively than the in vivo test; thus in vitro test shows that the DNA target of wild-type l-Crel may be the followings: gtc (recognized homing site in all the cited documents), gcc or gtt triplet at the positions - 5 to -3, in reference to SEQ ID NO:65.
  • Q26C/Y66R drive specific elimination of selected DNA targets in vivo and display shifted specificities of DNA binding and cleavage in vitro.
  • the overall result of the selection and characterization of enzyme point mutants against individual target site variants is both a shift and a broadening in binding specificity and in kinetics of substrate cleavage.
  • Each mutant displays a higher dissociation constant (lower affinity) against the original wild-type target site than does the wild-type enzyme, and each mutant displays a lower dissociation constant (higher affinity) against its novel target than does the wild-type enzyme.
  • the enzyme mutants display similar kinetics of substrate cleavage, with shifts and broadening in substrate preferences similar to those described for binding affinities.
  • - WO 2004/067736 describes a general method for producing a custom-made meganuclease able to cleave a targeted DNA sequence derived from an initial meganuclease. This general method comprises the steps of preparing a library of meganuclease variants and selecting the variants able to cleave the targeted DNA sequence.
  • ⁇ -Cre ⁇ meganuclease variants which do not cleave wild-type ⁇ -CreI homing sites have not been described; however, there is a need for such novel ⁇ -CreI meganuclease variants with such a "modified specificity", i.e. meganuclease able to cleave at least one homing site other than the homing sites cleaved by the wild-type I-
  • Such variants would be of a particular interest for genetic and genome engineering.
  • the inventors found that one mutation of at least one of the amino acid residues in positions 44, 68 and 70 of ⁇ -Crel is sufficient to obtain a ⁇ -Crel meganuclease variant able to cleave at least one homing site that is not cleaved by the wild-type meganuclease.
  • the subject-matter of the present invention is a method of preparing a ⁇ -CreI meganuclease variant having at least a DNA target sequence (or homing site) which is different from the homing sites of the wild type l-Crel meganuclease, said method comprising:
  • step (b) selecting the I-Crel meganuclease variants obtained in step (a) having at least one of the following R 3 triplet cleaving profile in reference to positions -5 Io -3 in a double-strand DNA target, said positions -5 to -3 corresponding to R 3 of the following formula I:
  • Ri is absent or present; and when present represents a nucleic acid fragment comprising 1 to 9 nucleotides corresponding either to a random nucleic acid sequence or to a fragment of a l-Crel meganuclease homing site situated from position -20 to -12 (from 5' to 3'), R] corresponding at least to position -12 of said homing site, R 2 represents the nucleic acid doublet ac or ct and corresponds to positions -7 to -6 of said homing site,
  • R 3 represents a nucleic acid triplet corresponding to said positions -5 to -3, selected among g, t, c and a, except the following triplets : gtc, gcc, gtg, gtt and get; therefore said nucleic acid triplet is preferably selected among the following triplets: ggg, gga, ggt, ggc, gag, gaa, gat, gac, gta, gcg, gca, tgg, tga, tgt, tgc, tag, taa, tat, tac, ttg, tta, ttt, ttc, teg, tea, tct, tec, agg, aga, agt, age, aag, aa, aat, aac, atg, ata, art, ate, acg, aca, act, ace, egg, c
  • R 4 represents the nucleic acid doublet gt or tc and corresponds to positions -2 to -1 of said homing site
  • R' i is absent or present; and when present represents a nucleic acid fragment comprising 1 to 9 nucleotides corresponding either to a random nucleic acid sequence or to a fragment of a I-Crel meganuclease homing site situated from position
  • R' 2 represents the nucleic acid doublet ag or gt, and corresponds to positions +6 to +7 of said homing site
  • R' 3 represents a nucleic acid triplet corresponding to said positions
  • R' 3 being different from gac, ggc, cac, aac, and age, when R 3 and R' 3 are non-palindromic, R' 4 represents the nucleic acid doublet gt or tc and corresponds to positions +1 to +2 of said homing site.
  • Glutamine residue means Arg or Arginine residue and D means Asp or Aspartic acid residue.
  • residue numbers refer to the amino acid numbering of the I-Crel sequence SWISSPROT P05725 or the pdb accession code Ig9y.
  • ADR l-Crel meganuclease in which amino acid residues Q44 and R68 have been replaced by alanine and aspartic acid, respectively, while R70 has not been replaced.
  • Other mutations that do not alter the cleavage activity of the variant are not indicated and the nomenclature adopted here does not limit the mutations to the only three posi- tions 44, 68 and 70.
  • nucleosides are designated as follows: one-letter code is used for designating the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine.
  • r represents g or a (purine nucleotides)
  • k represents g or t
  • s represents g or c
  • w represents a or t
  • m represents a or c
  • y represents t or c (pyrimidine nucleotides)
  • d represents g, a or t
  • v represents g, a or c
  • b represents g, t or c
  • h represents a, t or c
  • n represents g, a, t or c.
  • wild-type l-Crel designates a l-Crel meganuclase having the sequence SWISSPROT P05735 or pdb accession code Ig9y, and able to cleave the 24 bp double-strand polynucleotide sequence presented in figure 2B (positions -5 to -3: gtc) or double-strand polynucleotide sequences having at positions -5 to -3 the following other triplets: gtg, gtt, get or gcc.
  • wild-type 1-OeI cleaves not only homing sites which palindromic sequence in positions -5 to -3 is gtc, gcc or gtt (Seligman et al., 2002), but also gtg and get. It results that the variants of the invention are those able to cleave at least one homing site in which sequence in positions -5 to - 3 differs from gtc, get, gcc, gtt and gtg. Thus, wild-type 1-OeI is not only able to cleave homing site as described in figure 2B, i.e.
  • modified specificity relates to a l-Crel meganuclease variant able to cleave a homing site that is not cleaved, in the same conditions, by the wild-type ⁇ -CreI.
  • a ⁇ -Cre ⁇ meganuclease variant with a modified specificity is able to cleave at least one target site that is not cleaved by wild-type l-Crel.
  • heterodimeric form can be obtained for example by proceeding to the fusion of the two monomers.
  • Resulting heterodimeric meganuclease can be able to cleave at least one target site that is not cleaved by the homodimeric form. Therefore a meganuclease variant is still part of the invention when used in a heteromeric form.
  • the other monomer chosen for the formation of the heterodimeric meganuclease can be another variant monomer, but it can also be a wild-type monomer, for example a I- Crel monomer or a ⁇ -Dmo ⁇ monomer.
  • partially palindromic sequence is indiscrimi- nately used for designating a palindromic sequence having a broken symmetry.
  • 22 bp sequence Ci ia-ioa-9a-8a -7 c.
  • SEQ ID NO: 71 is a partially palindromic sequence in which symmetry is broken at base- pairs +/- 1 , 2, 6 and 7.
  • nucleotide sequences of positions +/- 8 to 1 1 and +/- 3 to 5 are palindromic sequences. Symmetry axe is situated between the base-pairs in positions -1 and +1.
  • targeting DNA construct corresponds to a DNA sequence comprising both the DNA target as defined hereabove and other DNA sequences allowing in vivo homologous recombination.
  • the inventors constructed a l-Crel variants library, each of them presenting at least one mutation in the amino acid residues in positions 44, 68 and/or 70 (pdb code Ig9y), and each of them being able to cleave at least one target site not cleaved by a wild-type I-Crel.
  • the mutation consists of the replacement of at least one amino acid residue in position 44, 68, and/or 70 by another residue selected in the group comprising A, D, E, G, H, K, N, P, Q, R, S and T.
  • Each mutated amino acid residue is changed independently from the other residues, and the selected amino acid residues can be the same or can be different from the other amino acid residues in position 44, 68 and/or 70.
  • the homing site cleaved by the 1-OeI meganuclease variant according to the invention but not cleaved by wild-type I- Crel, is the same as described above and illustrated in figure 2, except that the triplet sequence in positions -5 to -3 (corresponding to R 3 in formula I) and/or triplet sequence in positions +3 to +5 (corresponding to R 3 ' in formula I) differ from the triplet sequence in the same positions in the homing sites cleaved by the wild-type I- Cre ⁇ .
  • the ⁇ -Cre ⁇ meganuclease variants obtainable by the method described above, i.e. with a "modified specificity" are able to cleave at least one target that differs from wild-type 1-OeI target in positions -5 to -3 and/or in positions +3 to +5. It must be noted that said DNA target is not necessarily palin- dromic in positions +/- 3 to 5.
  • 1-OeI is active in homodimeric form, but may be active in a heterodimeric form.
  • 1-OeI variants according to the instant invention could be active not only in a homodimeric form, but also in a heterodimeric form, and in both cases, they could recognize a target with either palindromic or non palin- dromic sequence in position +/- 3 to 5, provided that the triplet in position -5 to -3 and/or +3 to +5 differs from gtc, gcc, gtg, gtt and get, and from gac, ggc, cac, aac, and age, respectively.
  • a variant able to cleave a plurality of targets could also cleave a target which sequence in position +/- 3 to 5 is not palindromic. Further, a variant could act both in a homodimeric form and in a heterodimeric form.
  • I-Crel variant could form a heterodimeric meganuclease, in which the other variant could be a wild-type ⁇ -Cre ⁇ monomer, another wild-type meganuclease monomer, such as ⁇ -Dmol, another ⁇ -Crel variant monomer, or a monomer of a variant from another meganuclease than l-Crel.
  • the other variant could be a wild-type ⁇ -Cre ⁇ monomer, another wild-type meganuclease monomer, such as ⁇ -Dmol, another ⁇ -Crel variant monomer, or a monomer of a variant from another meganuclease than l-Crel.
  • the I- Crel meganuclease variant obtained in step (b) is selected from the group consisting of: A44/A68/A70, A44/A68/G70, A44/A68/H70, A44/A68/K70, A44/A68/N70, A44/A68/Q70, A44/A68/R70, A44/A68/S70, A44/A68/T70, A44/D68/H70, A44/D68/K70, A44/D68/R70, A44/G68/H70, A44/G68/K70, A44/G68/N70, A44/G68/P70, A44/G68/R70, A44/H68/A70, A44/H68/G70, A44/H68/H70, A44/H68/K70, A44/H68/N70, A44/H68/Q70, A44/H68/R70, A44/A68/A70, A
  • E44/R68/A70 E44/R68/H70, E44/R68/N70, E44/R68/S70, E44/R68/T70,
  • G44/T68/D70 G44/T68/P70, G44/T68/R70, H44/A68/S70, H44/A68/T70, H44/R68/A70, H44/R68/D70, H44/R68/E70, H44/R68/G70, H44/R68/N70,
  • K44/R68/T70 K44/S68/A70, K44/S68/D70, K44/S68/H70, K44/S68/N70,
  • N44/P68/D70 N44/Q68/H70, N44/Q68/R70, N44/R68/A70, N44/R68/D70,
  • T44/A68/R70 T44/H68/R70, T44/K68/R70, T44/N68/P70, T44/N68/R70, T44/Q68/K70, T44/Q68/R70, T44/R68/A70, T44/R68/D70, T44/R68/E70, T44/R68/G70, T44/R68/H70, T44/R68/K70, T44/R68/N70, T44/R68/Q70, T44/R68/R70, T44/R68/S70, T44/R68/T70, T44/S68/K70, T44/S68/R70, T44/T68/K70, and T44/T68/R70.
  • the step (b) of selecting said l-Crel meganuclease variant is performed in vivo in yeast cells.
  • the subject-matter of the present invention is also the use of a ⁇ -CreI meganuclease variant as defined here above, i.e. obtainable by the method as described above, in vitro or in vivo for non-therapeutic purposes, for cleaving a double-strand nucleic acid target comprising at least a 20-24 bp partially palindromic sequence, wherein at least the sequence in positions +/- 8 to 11 is palindromic, and the nucleotide triplet in positions -5 to -3 and/or the nucleotide triplet in positions +3 to +5 differs from gtc, gcc, gtg, gtt, and get, and from gac, ggc, cac, aac and age, respectively.
  • Formula I describes such a DNA target.
  • said l-Crel meganuclease variant is selected from the group consisting of: A44/A68/A70, A44/A68/G70, A44/A68/H70, A44/A68/K70, A44/A68/N70, A44/A68/Q70, A44/A68/R70, A44/A68/S70, A44/A68/T70, A44/D68/H70, A44/D68/K70, A44/D68/R70, A44/G68/H70, A44/G68/K70, A44/G68/N70, A44/G68/P70, A44/G68/R70, A44/H68/A70, A44/H68/G70, A44/H68/H70, A44/H68/K70, A44/H68/N70, A44/H68/Q70, A44/H68/R70, A44/H68/R70, A44/H68
  • A44/Q68/S70 A44/R68/A70, A44/R68/D70, A44/R68/E70, A44/R68/G70,
  • G44/T68/P70 G44/T68/R70, H44/A68/S70, H44/A68/T70, H44/R68/A70,
  • K44/E68/S70 K44/G68/A70, K44/G68/G70, K44/G68/N70, K44/G68/S70,
  • K44/G68/T70 K44/H68/D70, K44/H68/E70, K44/H68/G70, K44/H68/N70, K44/H68/S70, K44/H68/T70, K44/K68/A70, K44/K68/D70, K44/K68/H70,
  • K44/K68/T70 K44/N68/A70, K44/N68/D70, K44/N68/E70, K44/N68/G70,
  • K44/Q68/T70 K44/R68/A70, K44/R68/D70, K44/R68/E70, K44/R68/G70, K44/R68/H70, K44/R68/N70, K44/R68/Q70, K44/R68/S70, K44/R68/T70,
  • R44/R68/Q70 R44/R68/S70, R44/R68/T70, R44/S68/G70, R44/S68/N70,
  • the I- Crel meganuclease variant is a homodimer.
  • said I- Crel meganuclease variant is a heterodimer. According to said use:
  • either the l-Crel meganuclease variant is able to cleave a DNA target in which sequence in positions +/- 3 to 5 is palindromic
  • said l-Crel meganuclease variant is able to cleave a DNA target in which sequence in positions +/- 3 to 5 is non-palindromic.
  • the cleaved nucleic acid target is a DNA target in which palindromic sequences in posi- tions -11 to -8 and +8 to +1 1 are caaa and tttg, respectively.
  • said I- Crel meganuclease variant further comprises a mutation in position 75, preferably said mutation is D75N or D75V.
  • said l-Crel meganuclease variant has an alanine (A), an aspartic acid (D) or a threonine (T) in position 44, for cleaving a DNA target comprising nucleotide A in position -4, and/or T in position +4.
  • said ⁇ -Cre ⁇ meganuclease variant has a lysine (K) or an arginine (R) in position 44, for cleaving a target comprising nucleotide C in position -4, and/or G in position +4.
  • the subject-matter of the present invention is also l-Crel meganuclease variants:
  • said l-Crel meganuclease variant according to the invention obtainable by the method as described above, has mainly a modified specificity, i.e. is able to cleave a DNA target that is not cleaved by wild-type l-Crel.
  • Such novel l-Crel meganucleases may be used either as very specific endonucleases in in vitro digestion, for restriction or mapping use, either in vivo or ex vivo as tools for genome engineering.
  • each one can be used as a new scaffold for a second round of mutagenesis and selection/screening, for the purpose of making novel, second generation homing endonucleases.
  • the l-Crel meganuclease variants according to the invention are mutated only at positions 44, 68 and/or 70 of the DNA binding domain.
  • the instant invention also includes different proteins able to form heterodimers: heterodimerization of two different proteins from the above list result also in cleavage of non palindromic sequences, made of two halves from the sites cleaved by the parental proteins alone. This can be obtained in vitro by adding the two different I- OeI variants in the reaction buffer, and in vivo or ex vivo by coexpression. Another possibility is to build a single-chain molecule, as described by Epinat et al. (Epinat et al., 2003). This single chain molecule would be the fusion of two different l-Crel variants, and should also result in the cleavage of chimeric, non-palindromic sequences.
  • the amino acid residue chosen for the replacement of the amino acid in positions 44, 68 and/or 70 is selected in the group comprising A, D, E, G, H, K, N, P, Q, R, S and T.
  • Said l-Crel meganuclease variant is able to cleave at least one target, as defined above, that is not cleaved by the wild-type VCr e ⁇ .
  • said ⁇ -Cre ⁇ meganuclease variant is selected in the group consisting of: A44/A68/A70, A44/A68/G70, A44/A68/H70, A44/A68/K70, A44/A68/N70, A44/A68/Q70, A44/A68/S70, A44/A68/T70, A44/D68/H70, A44/D68/K70, A44/D68/R70, A44/G68/H70, A44/G68/K70, A44/G68/N70, A44/G68/P70, A44/H68/A70, A44/H68/G70, A44/H68/H70, A44/H68/K70, A44/H68/N70, A44/H68/Q70, A44/H68/S70, A44/H68/T70, A44/K68/A70, A44/K68/G70,
  • G44/R68/Q70 G44/T68/D70, G44/T68/P70, G44/T68/R70, H44/A68/S70, H44/A68/T70, H44/R68/D70, H44/R68/E70, H44/R68/G70, H44/R68/N70,
  • K44/K68/D70 K44/K68/H70, K44/K68/T70, K44/N68/A70, K44/N68/D70,
  • the ⁇ -Cre ⁇ meganuclease variant has an alanine (A), an aspartic acid (D) or a threonine (T) in position 44 and cleaves a target comprising the nucleotide A in position -4, and/or T in position +4.
  • the I-Crel meganuclease variant of the invention has a lysine (K) or an arginine (R) in position 44 and cleaves a target comprising c in position -4, and/or g in position +4.
  • said l-Crel meganuclease variant may be a homodimer or a heterodimer. It may be able to cleave a palindromic or a non- palindromic DNA target. It may further comprise a mutation in position 75, as specified hereabove.
  • the subject-matter of the present invention is also a polynucleotide, characterized in that it encodes a ⁇ -Crel meganuclease variant according to the invention.
  • the subject-matter of the present invention is an expression cassette comprising said polynucleotide and regulation sequences such as a promoter, and an expression vector comprising said expression cassette.
  • the subject-matter of the present invention is also an expression vector, as described above, further comprising a targeting DNA construct.
  • said targeting DNA construct comprises a sequence sharing homologies with the region surrounding the cleavage site of the I-Crel meganuclease variant of the invention.
  • said targeting DNA construct comprises: a) sequences sharing homologies with the region surrounding the cleavage site of the l-Crel meganuclease variant according to claim, and b) sequences to be introduced flanked by sequence as in a).
  • the subject-matter of the present invention is also a cell, characterized in that it is modified by a polynucleotide as defined above or by a vector as defined above.
  • the subject-matter of the present invention is also a transgenic plant, characterized in that it comprises a polynucleotide as defined above, or a vector as defined above.
  • the subject-matter of the present invention is also a non-human transgenic mammal, characterized in that it comprises a polynucleotide as defined above or a vector as defined above.
  • the subject-matter of the present invention is further the use of a I- CVeI meganuclease variant, a polynucleotide, a vector, a cell, a transgenic plant, a non-human transgenic mammal, as defined above, for molecular biology, for in vivo or in vitro genetic engineering, and for in vivo or in vitro genome engineering, for non-therapeutic purposes.
  • Non therapeutic purposes include for example (i) gene targeting of specific loci in cell packaging lines for protein production, (ii) gene targeting of specific loci in crop plants, for strain improvements and metabolic engineering, (iii) targeted recombination for the removal of markers in genetically modified crop plants, (iv) targeted recombination for the removal of markers in genetically modified microorganism strains (for antibiotic production for example).
  • it is for inducing a double-strand break in a site of interest comprising a DNA target sequence, thereby inducing a DNA recombination event, a DNA loss or cell death.
  • said double-strand break is for: repairing a specific sequence, modifying a specific sequence, restoring a functional gene in place of a mutated one, attenuating or activating an endogenous gene of interest, introducing a mutation into a site of interest, introducing an exogenous gene or a part thereof, inactivating or detecting an endogenous gene or a part thereof, translocating a chromosomal arm, or leaving the DNA unrepaired and degraded.
  • said I- Crel meganuclease variant, polynucleotide, vector, cell, transgenic plant or non- human transgenic mammal are associated with a targeting DNA construct as defined above.
  • the subject-matter of the present invention is also a method of genetic engineering, characterized in that it comprises a step of double-strand nucleic acid breaking in a site of interest located on a vector, comprising a DNA target of a I-
  • Crel meganuclease variant as defined above, thereby inducing a homologous recom- bination with another vector presenting homology with the sequence surrounding the cleavage site of said ⁇ -Crel meganuclease variant.
  • the subjet-matter of the present invention is also a method of genome engineering, characterized in that it comprises the following steps: 1) double- strand breaking a genomic locus comprising at least one recognition and cleavage site of a l-Crel meganuclease variant as defined above, by contacting said cleavage site with said 1-OeI meganuclease variant; 2) maintaining said broken genomic locus under conditions appropriate for homologous recombination with a targeting DNA construct comprising the sequence to be introduced in said locus, flanked by sequences sharing homologies with the target locus.
  • the subjet-matter of the present invention is also a method of genome engineering, characterized in that it comprises the following steps: 1) double- strand breaking a genomic locus comprising at least one recognition and cleavage site of a l-Crel meganuclease variant as defined above, by contacting said cleavage site with said l-Crel meganuclease variant; 2) maintaining said broken genomic locus under conditions appropriate for homologous recombination with chromosomal DNA sharing homologies to regions surrounding the cleavage site.
  • the subject-matter of the present invention is also a composition characterized in that it comprises at least one 1-OeI meganuclease variant, a polynucleotide or a vector as defined above.
  • composition in a preferred embodiment, it comprises a targeting DNA construct comprising the sequence which repairs the site of interest flanked by sequences sharing homologies with the targeted locus.
  • the subject-matter of the present invention is also the use of at least one l-Crel meganuclease variant, a polynucleotide or a vector, as defined above for the preparation of a medicament for preventing, improving or curing a genetic disease in an individual in need thereof, said medicament being administrated by any means to said individual.
  • the subject-matter of the present invention is also the use of at least one l-Crel meganuclease variant, a polynucleotide or a vector as defined above for the preparation of a medicament for preventing, improving or curing a disease caused by an infectious agent that presents a DNA intermediate, in an individual in need thereof, said medicament being administrated by any means to said individual.
  • the subject-matter of the present invention is also the use of at least one 1-Crel meganuclease variant, a polynucleotide or a vector, as defined above, in vitro, for inhibiting the propagation, inactivating or deleting an infectious agent that presents a DNA intermediate, in biological derived products or products intended for biological uses or for disinfecting an object.
  • said infectious agent is a virus.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • a vector according to the present invention comprises, but is not limited to, a YAC (yeast artificial chromosome), a BAC (bacterial artificial), a baculovirus vector, a phage, a phagemid, a cosmid, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non chromosomal, semi-synthetic or synthetic DNA.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • plasmids refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • suitable vectors are known to those of skill in the art and commercially available, such as the following bacterial vectors: pQE7O, pQE6O, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174.
  • pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); P trc99a, pKK223-3, pKK233-3, pDR540, pRlT5 (Pharmacia); pWLNEO,pSV2CAT, pOG44, pXTI, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QlAexpress), pET (Novagen).
  • Figure 1 principle of the screening assay. Yeast are transformed with the meganuclease expressing vector, marked with the LEU2 gene, and individually mated with yeast transformed with the reporter plasmid, marked by the TRPl gene.
  • Two palindromic targets derived from the natural l-Crel target (here named C 1234, SEQ ID NO: 65).
  • the l-Crel natural target contains two palindromes, boxed in grey: the -8 to -12 and +8 to +12 nucleotides on one hand, and the -5 to -3 and +3 to +5 nucleotide on another hand.
  • Vertical dotted line, from which are numbered the nucleotide bases, represents the symmetry axe for the palindromic sequences.
  • From the natural target can be derived two palindromic sequences, C 1221 (SEQ ID NO: 12) and C4334 (SEQ ID NO:66). Both are cut by l-Crel, in vitro and in yeast. Only one strand of each target site is shown.
  • Arginine (R) residue in position 44 of a l-Crel monomer directly interacts with guanine in position -5 of the target sequence, while glutamine (Q) residue of position 44 and Arginine (R) residue of position 70 directly interact with adenine in position +4 and guanine in position +3 of the complementary strand, respectively.
  • the 64 targets are derived from the l-Crel natural target site (here, also named C 1221, SEQ ID NO: 12). They correspond to all the 24 bp palindromes resulting from substitutions at positions -5, -4, -3, +3, +4 and +5.
  • FIG 3 Nine examples of pattern.
  • Nine meganucleases are tested 4 times against the 64 targets described in Figure 2C.
  • the position of the different targets is indicated on the top, left panel.
  • Meganucleases are identified by the amino acids in positions 44, 68 and 70 (ex: KSS is K44, S68, S70, or K44/S68/S70). Numeration of the amino acids is according to pdb code Ig9y. QRR corresponds to the wild type (Q44/R68/R70).
  • the cleaved targets are indicated besides the panels.
  • Figure 4 cDNA sequence (SEQ ID NO: 69) used for obtaining the I-Crel N75 scaffold protein (SEQ ID NO: 70).
  • CDS is from base-pair 1 to base-pair 501 and the "STOP" codon TGA (not shown) follows the base-pair 501.
  • the protein further contains mutations that do not alter its activity; in the protein sequence (SEQ ID NO: 70), the two first N-terminal residues are methionine and alanine (MA), and the three C-terminal residues alanine, alanine and aspartic acid (AAD). These sequences allow having DNA coding sequences comprising the Ncol (ccatgg) and Eagl (cggccg) restriction sites, which are used for cloning into various vectors.
  • Figure 5 pCLS0542 expression vector for meganucleases.
  • the meganuclease expression vector is marked by LEU2.
  • cDNAs encoding 1-OeI meganuclease variants are cloned into this vector digested with Ncol and Eagl, in order to have the variant expression driven by the inducible Gal 10 promoter.
  • Figure 6 pCLS0042 reporter vector.
  • the reporter vector is marked by TRPl and URA3.
  • the LacZ tandem repeats share 800 bp of homology, and are separated by 1 ,3 kb of DNA. They are surrounded by ADH promoter and terminator sequences. Target sites are cloned into the Smal site.
  • Figure 7 shows the results with 292 1-OeI meganuclease variants with a "modified specificity".
  • Proteins are defined by the amino acid present in positions 44, 68 and 70 (three first columns). Numeration of the amino acids is according to pdb accession code Ig9y. Targets are defined by nucleotides at positions -5 to -3 . For each protein, observed cleavage (1) or non observed cleavage (0) is shown for each one of the 64 targets. Examples The following examples are presented here only for illustrating the invention and not for limiting the scope thereof. Other variants, obtained from a cDNA, which sequence differs from SEQ ID NO: 69, and using appropriate primers, are still part of the invention.
  • Example 1 Experimental procedure Construction of the library of the 1-OeI variants (Ulib2 library)
  • a combinatorial library was constructed by mutagenesis of the I- CVeI homing endonuclease replacing DNA binding residues. Three residues (Q44, R68 and R70) capable of specific interactions with three bases in a single half-site within the DNA target (Jurica et ai, 1998) were selected.
  • the combinatorial library was obtained by replacing the three corresponding codons with a unique degenerated vvk codon. vvk corresponds to 18 different codons coding for 12 different amino acids (A, D, E, G, H, K, N, P, Q, R, S and T), as a consequence of the degeneracy of the genetic code.
  • mutants in the protein library corresponded to independant combinations of any of the 12 amino acids encoded by the wk codon at three residue positions.
  • the maximal (theoretical) diversity of the protein library was 12 3 or 1728.
  • the diversity is 18 3 or 5832.
  • residue D75 which is shielded from solvent by R68 and R70, was mutated to N in order to remove the likely energetic strain caused by replacements of those two basic residues in the library.
  • Homodimers of mutant D75N purified from E. coli cells wherein it was over-expressed using a pET expression vector
  • D75N gene i.e. a wild-type l-Crel, which CDS is shown in figure
  • PCR reaction The conditions of the PCR reaction are as follows: plasmid pET24-T45 containing the gene I- CVeI D75N was diluted at 1 ng/ ⁇ l to be used as template for PCR. Degenerated oligonucleotides encoding the desired randomizations were used to amplify a PCR fragment in 50 ⁇ l PCR reactions. PCR products were pooled, EtOH precipitated and resuspended in 50 ⁇ l 10 mM Tris. PCR products were cloned by ligation into the D75N mutant gene, within a pET expression vector digested with specific restriction enzymes.
  • Digestion of vector and insert DNA were conducted in two steps (single enzyme digestion) between which the DNA sample was extracted (using classic phenolxhloroform.isoamylalcohol-based methods) and EtOH-precipitated. 10 ⁇ g of digested vector DNA were used for ligation, with a 5:1 excess of insert DNA. E coli TGl cells were transformed with the resulting vector by electroporation. To produce a number of cell clones above the theoretical diversity of the library, 6x10 4 clones were produced (35 times the diversity). Bacterial clones were scraped from plates and the corresponding plasmid vectors were extracted and purified.
  • the library was eventually recloned in the yeast pCLS0542 vector ( Figure 5), by sub-cloning a NcoI-EagJ DNA fragment containing the entire 1-Crel ORF of the Figure 4A (SEQ ID NO: 69) in which the stop codon TGA which follows the bp 501 is not shown into pCLS0542.
  • the 64 palindromic targets are described in Figure 2C (positions -5 to -3 and +3 to +5) (SEQ ID NO: 1 to SEQ ID NO: 64).
  • 64 couples of oligonucleotides were designed, corresponding to the two strands of the 64 DNA targets, with 12 pb of non palindromic extra sequence on each side, were annealed and cloned into a pGEM-T vector (Promega). Then, a PvuJI restriction fragment was excised from each one of the 64 pGEM-T-derived vector, and cloned into pCLS0042 ( Figure 6), resulting in 64 yeast reporter vectors. Steps of excision, digestion and ligation are performed using typical methods known by those skilled in the art. Insertion of the target sequence is made at the Smal site of pCLS0042.
  • A44/R68/L70 variant were transformed into strain FYC2-6A: alpha, trpl ⁇ 63, leu2 ⁇ l, his3 ⁇ 200.
  • the target plasmids were transformed into yeast strain FYBL2-7B: a, ura3 ⁇ 851, trpl ⁇ 63, leu2 ⁇ l, lys2 ⁇ 202.
  • yeast strain FYBL2-7B a, ura3 ⁇ 851, trpl ⁇ 63, leu2 ⁇ l, lys2 ⁇ 202.
  • a classical chemical/heat choc protocol can be used, and routinely gives 10 6 independent transformants per ⁇ g of DNA; transfor- mants were selected on leucine drop-out synthetic medium (Gietz and Woods, 2002). Screening l-Cre ⁇ variant clones as well as yeast reporter strains were stocked in glycerol (20%) stock and replicated in novel microplates.
  • Each reporter strain was spotted 13 824 times on a nylon membrane, and on each one of this spot was spotted one out of the 13 824 yeast clones expressing a variant meganuclease.
  • Membranes were laid on solid agarose YEPD rich medium, and incubated at 3O 0 C for one night, to allow mating.
  • membranes were laid on synthetic medium, lacking leucine and tryptophane, and with galactose (1%) as a carbon source, and incubated for five days at 37°C, to select for diploids, allow for meganuclease expression, reporter plasmid cleavage and recombination, and expression of beta-galactosidase.
  • membranes were laid on solid agarose medium with 0.02% X-GaI in 0.5 M Sodium Phosphate buffer, pH 7.0, 0.1% SDS, 6% Dimethyl Formamide (DMF), 7 mM beta-mercaptoethanol, 1% agarose, and incubated at 37°C, to monitor beta- galactosidase activity. Positive clones were identified after two days of incubation, according to staining. For secondary screening, the same procedure was followed with the 292 selected positives, except tKat each mutant was tested 4 times on the same membrane (see figure 7).
  • Example 2 Identification of l-Crel meganuclease variants with modified cleavage specificity.
  • the meganucleases expressed from a replicative vector can be tested for their ability to cleave a DNA target in yeast cells, when this DNA target is placed between two direct repeats in another replicative vector. Efficient cleavage of the DNA target induces homologous recombination of direct repeats, resulting in the restoration of a functional beta-galactosidase marker, which can be monitored by X-GaI staining.
  • This method is used herein to screen a library of ⁇ -Crel meganuclease variants with a collection of DNA targets, in order to identify novel I- Oel-derived meganucleases with altered or modified specificities.
  • the library of ⁇ -Crel meganuclease variants was made by mutagenesis of an I-Crel scaffold which residue 75 was replaced with an Asparagine (N). Positions 44, 68 and 70 were randomized, and the regular amino acids (Q44, R68 and R70) replaced with one out of 12 amino acids (A, D, E, G, H, K, N, P, Q, R, S, or T, see Example 1). The resulting library has a complexity of 1728 in terms of protein (5832 in terms of nucleic acids, see Example 3) and was cloned in a yeast replicative expression vector carrying a LEU2 auxotrophic marker gene.
  • This library was transformed into a Ieu2 mutant haploid yeast strain (FYC2-6A). 13 824 transformant (Leu + ) clones were individually picked in 96 wells microplates.
  • a series of 64 targets (SEQ ID NO: 1 to SEQ ID NO: 64) were derived from the ⁇ -Cre ⁇ natural target site (SEQ ID NO: 65). These targets are all palindromic in positions +/- 3 to 5 and +/- 8 to 11, and triplet sequence at positions -5 to -3 was randomised as shown in figure 2C.
  • the 64 targets were cloned in the appropriate yeast reporter vector (see Example 1), and transformed into an haploid strain (FYBL2-7B), resulting in 64 tester strains.
  • l-Crel meganuclease variants such as QKS and QRK.
  • QKS and QRK l-Crel meganuclease variants
  • a lot of l-Crel meganuclease variants display very different patterns.
  • cleavage of a unique sequence is observed.
  • I- OeI meganuclease variants DRK, RAT and THR are active on the ggg, get and gac targets, respectively, which were not cleaved by wild-type l-Crel ( Figure 3).
  • QAT and QAN both cleave gtt, one of the targets cleaved by l-Crel.
  • NAR cleaves two different targets, gac and tac, both uncut by l-Crel.
  • Other l-Crel meganuclease variants cleave efficiently a series of different targets, such as KSS (cleaves net, ncc, ttt, ttc, ctt and etc) and NRS (gag, gat, gac and gat).
  • KSS cleaves net, ncc, ttt, ttc, ctt and etc
  • NRS gag, gat, gac and gat.
  • 25 are not cleaved by any of the 292 variants and it is notable that the nna sequence (except gta and gca) and the ngy sequence (except ggt and ggc) remain uncut.
  • l-Crel meganuclease variants Different groups of l-Crel meganuclease variants emerge from these results, for example : - a group comprising 1-OeI meganuclease variants that cleave more targets than QRR, such as GTP or NRK, a group comprising l-Crel meganuclease variants that cleave less targets than QRR, such as TAR, a group comprising l-Crel meganuclease variants that cleave only one target, which is not cut by the "wild-type QRR", such as ADH, ADK, AGH, AGK, AHK, AQD, HTT, DRA, DRK, DRR, DRT, GRQ, GTR, NAH, NHN, NKG, NKH, NSG, NTH, RAG, RAT, RGT, RNT, RRN, RSS, RST, SHR, THR, TKR,
  • Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc Natl Acad Sci U S A, 95, 5172-5177.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention se rapporte à un procédé pour préparer des variants de méganucléase I-CreI présentant une spécificité modifiée, à savoir la capacité de cliver au moins un site de localisation n'étant pas clivé par I-CreI de type sauvage. Cette invention concerne également les variants de méganucléase I-CreI productibles au moyen de ce procédé, et l'utilisation de ces variants de méganucléase I-CreI pour cliver une nouvelle cible ADN, ou en ingénierie génétique et génomique à des fins non thérapeutiques. La présente invention concerne en outre des acides nucléiques codant ces variants, des cassettes d'expression comprenant ces acides nucléiques, des vecteurs comportant ces cassettes d'expression, ainsi que des cellules ou des organismes, des plantes ou des animaux exceptés des êtres humains qui sont transformés par lesdits vecteurs.
PCT/IB2005/000981 2005-03-15 2005-03-15 Variants de meganuclease i-crei presentant une specificite modifiee, leur procede de preparation, et leurs utilisations Ceased WO2006097784A1 (fr)

Priority Applications (20)

Application Number Priority Date Filing Date Title
PCT/IB2005/000981 WO2006097784A1 (fr) 2005-03-15 2005-03-15 Variants de meganuclease i-crei presentant une specificite modifiee, leur procede de preparation, et leurs utilisations
EP10004717A EP2327772A1 (fr) 2005-03-15 2006-03-15 Variantes de méganucléase I-crel avec spécificité modifiée, procédé de préparation et utilisations associées
DE602006014107T DE602006014107D1 (de) 2005-03-15 2006-03-15 I-crei-meganuklease-varianten mit modifizierter spezifität sowie verfahren zu ihrer herstellung und verwendung
CA2600033A CA2600033C (fr) 2005-03-15 2006-03-15 Variantes des meganucleases i-crei a specificite modifiee: procede de preparation et d'utilisation correspondants
PCT/IB2006/001271 WO2006097854A1 (fr) 2005-03-15 2006-03-15 Meganucleases heterodimeriques et utilisation de ces dernieres
AT06744673T ATE466933T1 (de) 2005-03-15 2006-03-15 I-crei-meganuklease-varianten mit modifizierter spezifität sowie verfahren zu ihrer herstellung und verwendung
EP10004689A EP2327771A1 (fr) 2005-03-15 2006-03-15 Variantes de méganucléase I-crel avec spécificité modifiée, procédé de préparation et utilisations associées
EP10004688A EP2327773A1 (fr) 2005-03-15 2006-03-15 Variantes de méganucléase I-crel avec spécificité modifiée, procédé de préparation et utilisations associées
DK06744673.2T DK1863909T3 (da) 2005-03-15 2006-03-15 I-CreI-meganukleasevarianter med modificeret specificitet, fremgangsmåde til fremstilling og anvendelser deraf
AU2006224248A AU2006224248B2 (en) 2005-03-15 2006-03-15 I-Crei meganuclease variants with modified specificity, method of preparation and uses thereof
JP2008501447A JP2008535484A (ja) 2005-03-15 2006-03-15 特異性が改変されたＩ−ＣｒｅＩメガヌクレアーゼ変異型、その作製方法及びその使用
ES06744673T ES2347684T3 (es) 2005-03-15 2006-03-15 Variantes de la meganucleasa i-crei con especificidad modifica, metodo de preparacion y usos de las mismas.
US11/908,934 US20110158974A1 (en) 2005-03-15 2006-03-15 Heterodimeric Meganucleases and Use Thereof
PCT/IB2006/001203 WO2006097853A1 (fr) 2005-03-15 2006-03-15 Variantes des meganucleases i-crei a specificite modifiee: procede de preparation et d'utilisation correspondants
US11/908,798 US7897372B2 (en) 2005-03-15 2006-03-15 I-CreI meganuclease variants with modified specificity, method of preparation and uses thereof
CN200680012709.7A CN101198694B (zh) 2005-03-15 2006-03-15 具有经修饰的特异性的I-CreI大范围核酸酶变体、其制备方法及应用
EP06744673.2A EP1863909B2 (fr) 2005-03-15 2006-03-15 Variantes des méganucléases i-crei à spécificité modifiée: procédé de préparation et utilisations de celles-ci
EP10004687A EP2325307A1 (fr) 2005-03-15 2006-03-15 Variantes de méganucléase I-crel avec spécificité modifiée, procédé de préparation et utilisations associées
US12/859,905 US20110072527A1 (en) 2005-03-15 2010-08-20 I-crei meganuclease variants with modified specificity, method of preparation and uses thereof
US13/422,902 US8715992B2 (en) 2005-03-15 2012-03-16 I-CreI meganuclease variants with modified specificity, method of preparation and uses thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IB2005/000981 WO2006097784A1 (fr) 2005-03-15 2005-03-15 Variants de meganuclease i-crei presentant une specificite modifiee, leur procede de preparation, et leurs utilisations

Publications (1)

Publication Number Publication Date
WO2006097784A1 true WO2006097784A1 (fr) 2006-09-21

Family

ID=35134262

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2005/000981 Ceased WO2006097784A1 (fr) 2005-03-15 2005-03-15 Variants de meganuclease i-crei presentant une specificite modifiee, leur procede de preparation, et leurs utilisations

Country Status (2)

Country Link
CN (1) CN101198694B (fr)
WO (1) WO2006097784A1 (fr)

Cited By (81)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007049095A1 (fr) * 2005-10-25 2007-05-03 Cellectis Variants d'endonuclease de liaison a laglidadg comprenant des mutations dans deux sous-domaines fonctionnels et leur utilisation
WO2007047859A3 (fr) * 2005-10-18 2007-11-15 Prec Biosciences Meganucleases conçues rationnellement possedant une specificite sequence modifiee et une affinite de liaison pour l'adn
WO2008010009A1 (fr) * 2006-07-18 2008-01-24 Cellectis Variants de méganucléase clivant une séquence d'adn cible provenant d'un gène rag et leurs utilisations
WO2008059317A1 (fr) * 2006-11-14 2008-05-22 Cellectis Variantes de méganucléases clivant une séquence cible d'adn du gène hprt et leurs utilisations
WO2008059382A3 (fr) * 2006-11-14 2008-08-07 Cellectis Variants de méganucléase clivant une séquence cible d'adn à partir du gène hprt et leurs utilisations
WO2008102198A1 (fr) 2007-02-19 2008-08-28 Cellectis Variants d'endonucléase homing laglidadg à une nouvelle spécificité de substrat et leur utilisation
WO2008102199A1 (fr) * 2007-02-20 2008-08-28 Cellectis Variants de méganucléase clivant une séquence cible d'adn provenant du gène de la bêta-2-microglobuline et utilisations de ceux-ci
WO2009019528A1 (fr) * 2007-08-03 2009-02-12 Cellectis Variants de méganucléases clivant une séquence cible d'adn provenant du gène de la chaine gamma du récepteur d'interleukine-2 humain et ses utilisations
WO2009095742A1 (fr) * 2008-01-31 2009-08-06 Cellectis Nouvelle méganucléase à chaîne unique dérivée de l'i-crei et ses utilisations
WO2010015899A3 (fr) * 2008-08-04 2010-03-25 Cellectis Nouvelle méthode pour générer des méganucléases ayant des caractéristiques modifiées
WO2010136981A2 (fr) 2009-05-26 2010-12-02 Cellectis Variants de méganucléase clivant le génome d'un virus non intégratif pathogène et leurs utilisations
WO2011007336A1 (fr) 2009-07-17 2011-01-20 Cellectis Vecteurs viraux codant pour une matrice de réparation d'adn et contenant une méganucléase spécifique de site associée à un virion pour un ciblage de gène
WO2011021166A1 (fr) 2009-08-21 2011-02-24 Cellectis Variants de méganucléase clivant une séquence d’adn cible du gène d’acide lysosomique alpha-glucosidase humain et utilisations de ceux-ci
US7959164B2 (en) 2007-03-23 2011-06-14 Arvinmeritor Technology, Llc Roll control devices
WO2011082310A2 (fr) 2009-12-30 2011-07-07 Pioneer Hi-Bred International, Inc. Procédés et compositions pour la modification ciblée de polynucléotides
WO2011101811A2 (fr) 2010-02-18 2011-08-25 Cellectis Système amélioré de recombinaison méganucléase
WO2011104382A1 (fr) 2010-02-26 2011-09-01 Cellectis Utilisation d'endonucléases pour insérer des transgènes dans des locus safe harbor
WO2011135407A1 (fr) * 2010-04-30 2011-11-03 Cellectis Variants de i-crei ayant une nouvelle spécificité et procédés pour leur génération
WO2011141825A1 (fr) 2010-05-12 2011-11-17 Cellectis Variants de méganucléase clivant une séquence cible d'adn du gène de la rhodopsine et utilisations correspondantes
WO2011141820A1 (fr) 2010-05-12 2011-11-17 Cellectis Variants de méganucléase clivant une séquence cible d'adn du gène de dystrophine et leurs utilisations
WO2012001527A2 (fr) 2010-06-15 2012-01-05 Cellectis S.A. Procédé permettant d'améliorer le clivage d'un adn par une endonucléase sensible à la méthylation
WO2012004671A2 (fr) 2010-07-07 2012-01-12 Cellectis Variants de méganucléases coupant une séquence cible d'adn dans le gène nanog et leurs utilisations
WO2012007848A2 (fr) 2010-07-16 2012-01-19 Cellectis Variants de méganucléase clivant une séquence d'adn cible dans le gène was et leurs utilisations
WO2012010976A2 (fr) 2010-07-15 2012-01-26 Cellectis Variants de méganucléases clivant une séquence d'adn cible dans le gène tert et leurs utilisations
WO2012058458A2 (fr) 2010-10-27 2012-05-03 Cellectis Sa Procédé permettant d'augmenter l'efficacité d'une mutagenèse induite par des cassures double brin
WO2012129373A2 (fr) 2011-03-23 2012-09-27 Pioneer Hi-Bred International, Inc. Procédés de production d'un locus complexe de caractéristique transgénique
WO2012138901A1 (fr) 2011-04-05 2012-10-11 Cellectis Sa Procédé pour l'amélioration de l'efficacité d'endonucléases à coupure rare et ses utilisations
WO2012138927A2 (fr) 2011-04-05 2012-10-11 Philippe Duchateau Procédé de génération de nucléases tale compactes et leurs utilisations
WO2012149470A1 (fr) 2011-04-27 2012-11-01 Amyris, Inc. Procédés de modification génomique
WO2012158778A1 (fr) 2011-05-16 2012-11-22 Cellectis S.A. Souches d'algues ou de cyanobactéries transgéniques deux fois plus sûres pour empêcher leur implantation et leur dissémination dans des écosystèmes naturels
EP2535406A1 (fr) 2007-07-23 2012-12-19 Cellectis Variantes de méganucléase clivant une séquence cible d'ADN provenant du gène bêta de l'hémoglobine humaine et leurs utilisations
US8338157B2 (en) 2008-03-11 2012-12-25 Precision Biosciences, Inc. Rationally-designed meganuclease variants of lig-34 and I-crei for maize genome engineering
WO2013009525A1 (fr) 2011-07-08 2013-01-17 Cellectis S.A. Procédé d'amélioration de l'efficacité de mutagenèse induite par une cassure double brin
WO2013019411A1 (fr) 2011-08-03 2013-02-07 E. I. Du Pont De Nemours And Company Procédés et compositions permettant une intégration ciblée dans une plante
EP2568048A1 (fr) 2007-06-29 2013-03-13 Pioneer Hi-Bred International, Inc. Procédés de modification du génome d'une cellule de plante monocotylédone
WO2013066423A2 (fr) 2011-06-21 2013-05-10 Pioneer Hi-Bred International, Inc. Procédés et compositions pour obtenir des plantes stériles mâles
US8445251B2 (en) 2007-10-31 2013-05-21 Precision Biosciences, Inc. Rationally-designed single-chain meganucleases with non-palindromic recognition sequences
US8450561B2 (en) 2008-02-29 2013-05-28 Monsanto Technology Llc Corn plant event MON87460 and compositions and methods for detection thereof
US8530214B2 (en) 2003-01-28 2013-09-10 Cellectis S.A. Use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof
WO2013166113A1 (fr) 2012-05-04 2013-11-07 E. I. Du Pont De Nemours And Company Compositions et procédés comprenant des séquences ayant une activité de méganucléase
EP2770052A1 (fr) 2007-06-06 2014-08-27 Cellectis Procédé d'amélioration de l'activité de clivage de méganucléases dérivées de I-creI
WO2014164466A1 (fr) 2013-03-12 2014-10-09 E. I. Du Pont De Nemours And Company Procédés d'identification de sites de reconnaissance de variant pour agents d'induction de cassure double brin à coupure rare, génétiquement modifiés, leurs compositions et leurs utilisations
EP2853598A1 (fr) 2013-09-25 2015-04-01 Board of Regents of the University of Nebraska Procédés et compositions permettant d'obtenir des caractéristiques de plantes utiles
WO2015088643A1 (fr) 2013-12-11 2015-06-18 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification ciblée d'un génome
WO2015095804A1 (fr) 2013-12-19 2015-06-25 Amyris, Inc. Procédés d'intégration génomique
WO2015188109A1 (fr) 2014-06-06 2015-12-10 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification d'un locus ciblé
WO2015200805A2 (fr) 2014-06-26 2015-12-30 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour modification génétiques ciblées et procédés d'utilisation
WO2016061374A1 (fr) 2014-10-15 2016-04-21 Regeneron Pharmaceuticals, Inc. Méthodes et compositions de production ou de conservation de cellules pluripotentes
WO2016100819A1 (fr) 2014-12-19 2016-06-23 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour modification génétique ciblée par ciblage multiple en une seule étape
EP2352821B1 (fr) * 2008-09-08 2016-11-23 Cellectis Variants de méganucléase clivant une séquence d'adn cible provenant d'un gène de la glutamine synthétase et leurs utilisations
US9683257B2 (en) 2008-07-14 2017-06-20 Precision Biosciences, Inc. Recognition sequences for I-CreI-derived meganucleases and uses thereof
WO2018023014A1 (fr) 2016-07-29 2018-02-01 Regeneron Pharmaceuticals, Inc. Souris comprenant des mutations entraînant l'expression de la fibrilline 1 tronquée en c
EP3354732A1 (fr) 2014-06-23 2018-08-01 Regeneron Pharmaceuticals, Inc. Assemblage d'adn mediée par nuclease
US10072069B2 (en) 2007-06-01 2018-09-11 Open Monoclonal Technology, Inc. Humanized monoclonal antibodies from a transgenic rat
WO2019043082A1 (fr) 2017-08-29 2019-03-07 Kws Saat Se Aleurone bleue améliorée et autres systèmes de ségrégation
EP3456831A1 (fr) 2013-04-16 2019-03-20 Regeneron Pharmaceuticals, Inc. Modification ciblée du génome d'un rat
EP3460063A1 (fr) 2013-12-11 2019-03-27 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification ciblée d'un génome
WO2019067875A1 (fr) 2017-09-29 2019-04-04 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus ttr humanisé et procédés d'utilisation
WO2019177978A1 (fr) 2018-03-12 2019-09-19 Pioneer Hi-Bred International, Inc. Utilisation de facteurs morphogéniques pour l'amélioration de l'édition génique
EP3560329A1 (fr) 2011-05-02 2019-10-30 Board of Regents of the University of Nebraska Plantes présentant des caractéristiques utiles et procédés associés
EP3561050A1 (fr) 2013-02-20 2019-10-30 Regeneron Pharmaceuticals, Inc. Modification génétique de rats
WO2020123377A1 (fr) 2018-12-10 2020-06-18 Neoimmunetech, Inc. Cellules défaillantes nrf-2 et utilisations de celles-ci
WO2020131632A1 (fr) 2018-12-20 2020-06-25 Regeneron Pharmaceuticals, Inc. Expansion de répétition à médiation par nucléase
WO2020206134A1 (fr) 2019-04-04 2020-10-08 Regeneron Pharmaceuticals, Inc. Procédés pour l'introduction sans cicatrice de modifications ciblées dans des vecteurs de ciblage
WO2020206139A1 (fr) 2019-04-04 2020-10-08 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus facteur 12 de coagulation humanisé
WO2020247452A1 (fr) 2019-06-04 2020-12-10 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus ttr humanisé ayant une mutation bêta-slip et procédés d'utilisation
WO2020247812A1 (fr) 2019-06-07 2020-12-10 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus d'albumine humanisé
WO2021048291A1 (fr) * 2019-09-12 2021-03-18 Glaxosmithkline Intellectual Property Development Limited Procédé de criblage de bibliothèques
WO2021108363A1 (fr) 2019-11-25 2021-06-03 Regeneron Pharmaceuticals, Inc. Régulation à la hausse médiée par crispr/cas d'un allèle ttr humanisé
WO2022240846A1 (fr) 2021-05-10 2022-11-17 Sqz Biotechnologies Company Méthodes pour administrer des molécules d'édition génomique au noyau ou au cytosol d'une cellule et leurs utilisations
WO2022251644A1 (fr) 2021-05-28 2022-12-01 Lyell Immunopharma, Inc. Cellules immunitaires déficientes en nr4a3 et leurs utilisations
WO2022256437A1 (fr) 2021-06-02 2022-12-08 Lyell Immunopharma, Inc. Cellules immunitaires déficientes en nr4a3 et leurs utilisations
WO2023064924A1 (fr) 2021-10-14 2023-04-20 Codiak Biosciences, Inc. Cellules productrices modifiées pour la production de vésicules extracellulaires
WO2023129974A1 (fr) 2021-12-29 2023-07-06 Bristol-Myers Squibb Company Génération de lignées de cellules avec site d'intégration
EP4219731A2 (fr) 2016-05-18 2023-08-02 Amyris, Inc. Compositions et procédés d'intégration génomique d'acides nucléiques dans des tampons d'atterrissage exogènes
WO2023220603A1 (fr) 2022-05-09 2023-11-16 Regeneron Pharmaceuticals, Inc. Vecteurs et procédés de production d'anticorps in vivo
WO2023225665A1 (fr) 2022-05-19 2023-11-23 Lyell Immunopharma, Inc. Polynucléotides ciblant nr4a3 et leurs utilisations
EP4291663A2 (fr) 2021-02-09 2023-12-20 The Regents Of The University Of California Régulateurs immunitaires impliqués dans la défense contre des maladies des plantes provoquées par des espèces de liberibacter
WO2024064958A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a
WO2024064952A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a surexprimant c-jun
US12016313B2 (en) 2017-01-19 2024-06-25 Omniab Operations, Inc. Human antibodies from transgenic rodents with multiple heavy chain immunoglobulin loci

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067736A2 (fr) * 2003-01-28 2004-08-12 Cellectis Meganuclease sur mesure et son utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067736A2 (fr) * 2003-01-28 2004-08-12 Cellectis Meganuclease sur mesure et son utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SELIGMAN L M ET AL: "Mutations altering the cleavage specificity of a homing endonuclease", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 30, no. 17, 1 September 2002 (2002-09-01), pages 3870 - 3879, XP002282592, ISSN: 0305-1048 *
SUSSMAN D ET AL: "Isolation and Characterization of New Homing Endonuclease Specificities at Individual Target Site Positions", JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 342, no. 1, 3 September 2004 (2004-09-03), pages 31 - 41, XP004844889, ISSN: 0022-2836 *

Cited By (148)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8624000B2 (en) 2003-01-28 2014-01-07 Cellectis S.A. Use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof
US8697395B2 (en) 2003-01-28 2014-04-15 Cellectis S.A. Use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof
US8530214B2 (en) 2003-01-28 2013-09-10 Cellectis S.A. Use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof
US8163514B2 (en) 2005-10-18 2012-04-24 Duke University Methods of cleaving DNA with rationally-designed meganucleases
US8119381B2 (en) 2005-10-18 2012-02-21 Duke University Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity
US8119361B2 (en) 2005-10-18 2012-02-21 Duke University Methods of cleaving DNA with rationally-designed meganucleases
US8124369B2 (en) 2005-10-18 2012-02-28 Duke University Method of cleaving DNA with rationally-designed meganucleases
US8129134B2 (en) 2005-10-18 2012-03-06 Duke University Methods of cleaving DNA with rationally-designed meganucleases
US8133697B2 (en) 2005-10-18 2012-03-13 Duke University Methods of cleaving DNA with rationally-designed meganucleases
US8143015B2 (en) 2005-10-18 2012-03-27 Duke University Methods of cleaving DNA with rationally-designed meganucleases
EP2650366A3 (fr) * 2005-10-18 2014-02-12 Precision Biosciences Meganucleases concues rationnellement possedant une specificité sequence modifiée et une affinité de liaison pour l'ADN
US8304222B1 (en) 2005-10-18 2012-11-06 Duke University Rationally-designed meganucleases with altered sequence specificity and heterodimer formation
WO2007047859A3 (fr) * 2005-10-18 2007-11-15 Prec Biosciences Meganucleases conçues rationnellement possedant une specificite sequence modifiee et une affinite de liaison pour l'adn
US8143016B2 (en) 2005-10-18 2012-03-27 Duke University Methods of cleaving DNA with rationally-designed meganucleases
US8148098B2 (en) 2005-10-18 2012-04-03 Duke University Methods of cleaving DNA with rationally-designed meganucleases
US8021867B2 (en) 2005-10-18 2011-09-20 Duke University Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity
US8377674B2 (en) 2005-10-18 2013-02-19 Duke University Method for producing genetically-modified cells with rationally-designed meganucleases with altered sequence specificity
EP2343369A1 (fr) * 2005-10-25 2011-07-13 Cellectis Variants d'endonucléase laglidadg présentant des mutations dans deux sous-domaines fonctionnels et leur utilisation
WO2007049095A1 (fr) * 2005-10-25 2007-05-03 Cellectis Variants d'endonuclease de liaison a laglidadg comprenant des mutations dans deux sous-domaines fonctionnels et leur utilisation
WO2007057781A3 (fr) * 2005-10-25 2007-10-04 Cellectis Variantes de l’endonuclease ciblee laglidadg presentant des mutations dans deux sous-domaines fonctionnels et leur utilisation.
EP2365066A1 (fr) * 2005-10-25 2011-09-14 Cellectis Variants d'endonucleases laglidadg comprenant des mutations dans deux sous-domaines fonctionnels et leur utilisation
EP2343368A1 (fr) * 2005-10-25 2011-07-13 Cellectis Variants d'endonucléases laglidadg comprenant des mutations dans deux sous-domaines fonctionnels et leur utilisation
WO2008010093A3 (fr) * 2006-07-18 2008-08-28 Cellectis Variants de méganucléases clivant une séquence d'adn cible d'un gène rag et leurs utilisations
WO2008010009A1 (fr) * 2006-07-18 2008-01-24 Cellectis Variants de méganucléase clivant une séquence d'adn cible provenant d'un gène rag et leurs utilisations
WO2008059317A1 (fr) * 2006-11-14 2008-05-22 Cellectis Variantes de méganucléases clivant une séquence cible d'adn du gène hprt et leurs utilisations
WO2008059382A3 (fr) * 2006-11-14 2008-08-07 Cellectis Variants de méganucléase clivant une séquence cible d'adn à partir du gène hprt et leurs utilisations
AU2007347328B2 (en) * 2007-02-19 2013-03-07 Cellectis LAGLIDADG homing endonuclease variants having novel substrate specificity and use thereof
WO2008102198A1 (fr) 2007-02-19 2008-08-28 Cellectis Variants d'endonucléase homing laglidadg à une nouvelle spécificité de substrat et leur utilisation
WO2008102274A3 (fr) * 2007-02-20 2009-02-12 Cellectis Variants de méganucléase clivant une séquence d'adn cible du gène de la bêta-2-microglobuline et leurs utilisations
WO2008102199A1 (fr) * 2007-02-20 2008-08-28 Cellectis Variants de méganucléase clivant une séquence cible d'adn provenant du gène de la bêta-2-microglobuline et utilisations de ceux-ci
US7959164B2 (en) 2007-03-23 2011-06-14 Arvinmeritor Technology, Llc Roll control devices
US10072069B2 (en) 2007-06-01 2018-09-11 Open Monoclonal Technology, Inc. Humanized monoclonal antibodies from a transgenic rat
EP2770052A1 (fr) 2007-06-06 2014-08-27 Cellectis Procédé d'amélioration de l'activité de clivage de méganucléases dérivées de I-creI
US8912392B2 (en) 2007-06-29 2014-12-16 Pioneer Hi-Bred International, Inc. Methods for altering the genome of a monocot plant cell
EP2568048A1 (fr) 2007-06-29 2013-03-13 Pioneer Hi-Bred International, Inc. Procédés de modification du génome d'une cellule de plante monocotylédone
EP2535406A1 (fr) 2007-07-23 2012-12-19 Cellectis Variantes de méganucléase clivant une séquence cible d'ADN provenant du gène bêta de l'hémoglobine humaine et leurs utilisations
WO2009019528A1 (fr) * 2007-08-03 2009-02-12 Cellectis Variants de méganucléases clivant une séquence cible d'adn provenant du gène de la chaine gamma du récepteur d'interleukine-2 humain et ses utilisations
WO2009019614A3 (fr) * 2007-08-03 2009-11-19 Cellectis Variants de méganucléase clivant une séquence cible d'adn provenant du gène de la chaîne gamma du récepteur de l'interleukine-2 humain, et leurs utilisations
US12351839B2 (en) 2007-10-31 2025-07-08 Precision Biosciences, Inc. Rationally-designed single-chain meganucleases with non-palindromic recognition sequences
US10041053B2 (en) 2007-10-31 2018-08-07 Precision Biosciences, Inc. Rationally-designed single-chain meganucleases with non-palindromic recognition sequences
US9434931B2 (en) 2007-10-31 2016-09-06 Precision Biosciences, Inc. Rationally-designed single-chain meganucleases with non-palindromic recognition sequences
US9340777B2 (en) 2007-10-31 2016-05-17 Precision Biosciences, Inc. Rationally-designed single-chain meganucleases with non-palindromic recognition sequences
EP3098309A1 (fr) 2007-10-31 2016-11-30 Precision Biosciences, Inc. Méganucléases monocaténaires concues rationnellement contenant des séquences de reconnaissance non palindromiques
US8445251B2 (en) 2007-10-31 2013-05-21 Precision Biosciences, Inc. Rationally-designed single-chain meganucleases with non-palindromic recognition sequences
EP2660317A1 (fr) 2007-10-31 2013-11-06 Precision Biosciences, Inc. Méganucléases monocaténaires concues rationnellement contenant des séquences de reconnaissance non palindromiques
US8927247B2 (en) 2008-01-31 2015-01-06 Cellectis, S.A. I-CreI derived single-chain meganuclease and uses thereof
WO2009095793A1 (fr) 2008-01-31 2009-08-06 Cellectis Nouvelle méganucléase à chaîne simple dérivée de i-crei et ses utilisations
WO2009095742A1 (fr) * 2008-01-31 2009-08-06 Cellectis Nouvelle méganucléase à chaîne unique dérivée de l'i-crei et ses utilisations
US10428345B2 (en) 2008-02-29 2019-10-01 Monsanto Technology Llc Corn plant event MON87460 and compositions and methods for detection thereof
US10851385B2 (en) 2008-02-29 2020-12-01 Monanto Technology Llc Corn plant event MON87460 and compositions and methods for detection thereof
US10100328B2 (en) 2008-02-29 2018-10-16 Monsanto Technology Llc Corn plant event MON87460 and compositions and methods for detection thereof
EP2602325A1 (fr) 2008-02-29 2013-06-12 Monsanto Technology LLC Plantes de maïs de la lignée MON87460, compositions et procédés de détection correspondants
US9228197B2 (en) 2008-02-29 2016-01-05 Monsanto Technology Llc Corn plant event MON87460 and compositions and methods for detection thereof
US8450561B2 (en) 2008-02-29 2013-05-28 Monsanto Technology Llc Corn plant event MON87460 and compositions and methods for detection thereof
US12134774B2 (en) 2008-02-29 2024-11-05 Monsanto Technology Llc Corn plant event MON87460 and compositions and methods for detection thereof
US8338157B2 (en) 2008-03-11 2012-12-25 Precision Biosciences, Inc. Rationally-designed meganuclease variants of lig-34 and I-crei for maize genome engineering
US10287626B2 (en) 2008-07-14 2019-05-14 Precision Biosciences, Inc. Recognition sequences for I-CreI-derived meganucleases and uses thereof
EP4001408A2 (fr) 2008-07-14 2022-05-25 Precision Biosciences, Inc. Séquences de reconnaissance de méganucléases dérivées d'i-crei et utilisations associées
US9683257B2 (en) 2008-07-14 2017-06-20 Precision Biosciences, Inc. Recognition sequences for I-CreI-derived meganucleases and uses thereof
EP3211075A2 (fr) 2008-07-14 2017-08-30 Precision Biosciences, Inc. Séquences de reconnaissance de méganucléases dérivées d'i-crel et utilisations associées
EP3495478A2 (fr) 2008-07-14 2019-06-12 Precision Biosciences, Inc. Séquences de reconnaissance de méganucléases dérivées d'i-crel et utilisations associées
US10273524B2 (en) 2008-07-14 2019-04-30 Precision Biosciences, Inc. Recognition sequences for I-CreI-derived meganucleases and uses thereof
WO2010015899A3 (fr) * 2008-08-04 2010-03-25 Cellectis Nouvelle méthode pour générer des méganucléases ayant des caractéristiques modifiées
EP2352821B1 (fr) * 2008-09-08 2016-11-23 Cellectis Variants de méganucléase clivant une séquence d'adn cible provenant d'un gène de la glutamine synthétase et leurs utilisations
WO2010136981A2 (fr) 2009-05-26 2010-12-02 Cellectis Variants de méganucléase clivant le génome d'un virus non intégratif pathogène et leurs utilisations
WO2011007336A1 (fr) 2009-07-17 2011-01-20 Cellectis Vecteurs viraux codant pour une matrice de réparation d'adn et contenant une méganucléase spécifique de site associée à un virion pour un ciblage de gène
WO2011021166A1 (fr) 2009-08-21 2011-02-24 Cellectis Variants de méganucléase clivant une séquence d’adn cible du gène d’acide lysosomique alpha-glucosidase humain et utilisations de ceux-ci
WO2011082310A2 (fr) 2009-12-30 2011-07-07 Pioneer Hi-Bred International, Inc. Procédés et compositions pour la modification ciblée de polynucléotides
WO2011101811A2 (fr) 2010-02-18 2011-08-25 Cellectis Système amélioré de recombinaison méganucléase
WO2011101696A1 (fr) 2010-02-18 2011-08-25 Cellectis Système de recombinaison de méganucléase amélioré
US9290748B2 (en) 2010-02-26 2016-03-22 Cellectis Use of endonucleases for inserting transgenes into safe harbor loci
WO2011104382A1 (fr) 2010-02-26 2011-09-01 Cellectis Utilisation d'endonucléases pour insérer des transgènes dans des locus safe harbor
WO2011135407A1 (fr) * 2010-04-30 2011-11-03 Cellectis Variants de i-crei ayant une nouvelle spécificité et procédés pour leur génération
WO2011141825A1 (fr) 2010-05-12 2011-11-17 Cellectis Variants de méganucléase clivant une séquence cible d'adn du gène de la rhodopsine et utilisations correspondantes
WO2011141820A1 (fr) 2010-05-12 2011-11-17 Cellectis Variants de méganucléase clivant une séquence cible d'adn du gène de dystrophine et leurs utilisations
WO2012001527A2 (fr) 2010-06-15 2012-01-05 Cellectis S.A. Procédé permettant d'améliorer le clivage d'un adn par une endonucléase sensible à la méthylation
WO2012004671A3 (fr) * 2010-07-07 2012-06-14 Cellectis Variants de méganucléases coupant une séquence cible d'adn dans le gène nanog et leurs utilisations
WO2012004671A2 (fr) 2010-07-07 2012-01-12 Cellectis Variants de méganucléases coupant une séquence cible d'adn dans le gène nanog et leurs utilisations
WO2012010976A3 (fr) * 2010-07-15 2012-08-02 Cellectis Variants de méganucléases clivant une séquence d'adn cible dans le gène tert et leurs utilisations
WO2012010976A2 (fr) 2010-07-15 2012-01-26 Cellectis Variants de méganucléases clivant une séquence d'adn cible dans le gène tert et leurs utilisations
WO2012007848A2 (fr) 2010-07-16 2012-01-19 Cellectis Variants de méganucléase clivant une séquence d'adn cible dans le gène was et leurs utilisations
WO2012058458A2 (fr) 2010-10-27 2012-05-03 Cellectis Sa Procédé permettant d'augmenter l'efficacité d'une mutagenèse induite par des cassures double brin
WO2012129373A2 (fr) 2011-03-23 2012-09-27 Pioneer Hi-Bred International, Inc. Procédés de production d'un locus complexe de caractéristique transgénique
US9315788B2 (en) 2011-04-05 2016-04-19 Cellectis, S.A. Method for the generation of compact TALE-nucleases and uses thereof
WO2012138901A1 (fr) 2011-04-05 2012-10-11 Cellectis Sa Procédé pour l'amélioration de l'efficacité d'endonucléases à coupure rare et ses utilisations
EP3320910A1 (fr) 2011-04-05 2018-05-16 Cellectis Procédé de génération de nucléases tale compactes et leurs utilisations
WO2012138927A2 (fr) 2011-04-05 2012-10-11 Philippe Duchateau Procédé de génération de nucléases tale compactes et leurs utilisations
US11198856B2 (en) 2011-04-05 2021-12-14 Cellectis Method for the generation of compact tale-nucleases and uses thereof
US8685737B2 (en) 2011-04-27 2014-04-01 Amyris, Inc. Methods for genomic modification
WO2012149470A1 (fr) 2011-04-27 2012-11-01 Amyris, Inc. Procédés de modification génomique
US9701971B2 (en) 2011-04-27 2017-07-11 Amyris, Inc. Methods for genomic modification
EP3560329A1 (fr) 2011-05-02 2019-10-30 Board of Regents of the University of Nebraska Plantes présentant des caractéristiques utiles et procédés associés
WO2012158778A1 (fr) 2011-05-16 2012-11-22 Cellectis S.A. Souches d'algues ou de cyanobactéries transgéniques deux fois plus sûres pour empêcher leur implantation et leur dissémination dans des écosystèmes naturels
US9574208B2 (en) 2011-06-21 2017-02-21 Ei Du Pont De Nemours And Company Methods and compositions for producing male sterile plants
WO2013066423A2 (fr) 2011-06-21 2013-05-10 Pioneer Hi-Bred International, Inc. Procédés et compositions pour obtenir des plantes stériles mâles
WO2013009525A1 (fr) 2011-07-08 2013-01-17 Cellectis S.A. Procédé d'amélioration de l'efficacité de mutagenèse induite par une cassure double brin
WO2013019411A1 (fr) 2011-08-03 2013-02-07 E. I. Du Pont De Nemours And Company Procédés et compositions permettant une intégration ciblée dans une plante
WO2013166113A1 (fr) 2012-05-04 2013-11-07 E. I. Du Pont De Nemours And Company Compositions et procédés comprenant des séquences ayant une activité de méganucléase
US9909110B2 (en) 2012-05-04 2018-03-06 E. I. Du Pont De Nemours And Company Compositions and methods comprising sequences having meganuclease activity
US9499827B2 (en) 2012-05-04 2016-11-22 E I Du Pont De Nemours And Company Compositions and methods comprising sequences having meganuclease activity
US10150956B2 (en) 2012-05-04 2018-12-11 E I Du Pont De Nemours And Company Compositions and methods comprising sequences having meganuclease activity
EP3561050A1 (fr) 2013-02-20 2019-10-30 Regeneron Pharmaceuticals, Inc. Modification génétique de rats
WO2014164466A1 (fr) 2013-03-12 2014-10-09 E. I. Du Pont De Nemours And Company Procédés d'identification de sites de reconnaissance de variant pour agents d'induction de cassure double brin à coupure rare, génétiquement modifiés, leurs compositions et leurs utilisations
US10329574B2 (en) 2013-03-12 2019-06-25 E I Du Pont De Nemours And Company Methods for the identification of variant recognition sites for rare-cutting engineered double-strand-break-inducing agents and compositions and uses thereof
EP3456831A1 (fr) 2013-04-16 2019-03-20 Regeneron Pharmaceuticals, Inc. Modification ciblée du génome d'un rat
EP2853598A1 (fr) 2013-09-25 2015-04-01 Board of Regents of the University of Nebraska Procédés et compositions permettant d'obtenir des caractéristiques de plantes utiles
WO2015088643A1 (fr) 2013-12-11 2015-06-18 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification ciblée d'un génome
EP3460063A1 (fr) 2013-12-11 2019-03-27 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification ciblée d'un génome
EP4349980A2 (fr) 2013-12-11 2024-04-10 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification ciblée d'un génome
WO2015095804A1 (fr) 2013-12-19 2015-06-25 Amyris, Inc. Procédés d'intégration génomique
WO2015188109A1 (fr) 2014-06-06 2015-12-10 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification d'un locus ciblé
EP3708671A1 (fr) 2014-06-06 2020-09-16 Regeneron Pharmaceuticals, Inc. Procédés et compositions permettant la modification d'un locus cible
EP3708663A1 (fr) 2014-06-23 2020-09-16 Regeneron Pharmaceuticals, Inc. Assemblage d'adn mediée par nuclease
EP3354732A1 (fr) 2014-06-23 2018-08-01 Regeneron Pharmaceuticals, Inc. Assemblage d'adn mediée par nuclease
WO2015200805A2 (fr) 2014-06-26 2015-12-30 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour modification génétiques ciblées et procédés d'utilisation
EP3461885A1 (fr) 2014-06-26 2019-04-03 Regeneron Pharmaceuticals, Inc. Procèdes et compositions pour la modification génétique ciblé et procèdes d'utilisation
EP3561052A1 (fr) 2014-10-15 2019-10-30 Regeneron Pharmaceuticals, Inc. Méthodes et compositions de production ou de conservation de cellules pluripotentes
WO2016061374A1 (fr) 2014-10-15 2016-04-21 Regeneron Pharmaceuticals, Inc. Méthodes et compositions de production ou de conservation de cellules pluripotentes
EP3653048A1 (fr) 2014-12-19 2020-05-20 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour modification génétique ciblée par ciblage multiple en une seule étape
WO2016100819A1 (fr) 2014-12-19 2016-06-23 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour modification génétique ciblée par ciblage multiple en une seule étape
EP4219731A2 (fr) 2016-05-18 2023-08-02 Amyris, Inc. Compositions et procédés d'intégration génomique d'acides nucléiques dans des tampons d'atterrissage exogènes
WO2018023014A1 (fr) 2016-07-29 2018-02-01 Regeneron Pharmaceuticals, Inc. Souris comprenant des mutations entraînant l'expression de la fibrilline 1 tronquée en c
US12016313B2 (en) 2017-01-19 2024-06-25 Omniab Operations, Inc. Human antibodies from transgenic rodents with multiple heavy chain immunoglobulin loci
US11697822B2 (en) 2017-08-29 2023-07-11 KWS SAAT SE & Co. KGaA Blue aleurone and other segregation systems
EP4591703A2 (fr) 2017-08-29 2025-07-30 KWS SAAT SE & Co. KGaA Ameliorations apportees a l'aleurone bleue et a d'autres systemes de segregation
EP4591704A2 (fr) 2017-08-29 2025-07-30 KWS SAAT SE & Co. KGaA Ameliorations apportees a l'aleurone bleue et a d'autres systemes de segregation
WO2019043082A1 (fr) 2017-08-29 2019-03-07 Kws Saat Se Aleurone bleue améliorée et autres systèmes de ségrégation
EP4276185A2 (fr) 2017-09-29 2023-11-15 Regeneron Pharmaceuticals, Inc. Rongeurs comprenant un locus ttr humanisé et procédés d'utilisation
WO2019067875A1 (fr) 2017-09-29 2019-04-04 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus ttr humanisé et procédés d'utilisation
WO2019177978A1 (fr) 2018-03-12 2019-09-19 Pioneer Hi-Bred International, Inc. Utilisation de facteurs morphogéniques pour l'amélioration de l'édition génique
WO2020123377A1 (fr) 2018-12-10 2020-06-18 Neoimmunetech, Inc. Cellules défaillantes nrf-2 et utilisations de celles-ci
WO2020131632A1 (fr) 2018-12-20 2020-06-25 Regeneron Pharmaceuticals, Inc. Expansion de répétition à médiation par nucléase
WO2020206134A1 (fr) 2019-04-04 2020-10-08 Regeneron Pharmaceuticals, Inc. Procédés pour l'introduction sans cicatrice de modifications ciblées dans des vecteurs de ciblage
WO2020206139A1 (fr) 2019-04-04 2020-10-08 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus facteur 12 de coagulation humanisé
WO2020247452A1 (fr) 2019-06-04 2020-12-10 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus ttr humanisé ayant une mutation bêta-slip et procédés d'utilisation
WO2020247812A1 (fr) 2019-06-07 2020-12-10 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus d'albumine humanisé
WO2021048291A1 (fr) * 2019-09-12 2021-03-18 Glaxosmithkline Intellectual Property Development Limited Procédé de criblage de bibliothèques
WO2021108363A1 (fr) 2019-11-25 2021-06-03 Regeneron Pharmaceuticals, Inc. Régulation à la hausse médiée par crispr/cas d'un allèle ttr humanisé
EP4291663A2 (fr) 2021-02-09 2023-12-20 The Regents Of The University Of California Régulateurs immunitaires impliqués dans la défense contre des maladies des plantes provoquées par des espèces de liberibacter
WO2022240846A1 (fr) 2021-05-10 2022-11-17 Sqz Biotechnologies Company Méthodes pour administrer des molécules d'édition génomique au noyau ou au cytosol d'une cellule et leurs utilisations
WO2022251644A1 (fr) 2021-05-28 2022-12-01 Lyell Immunopharma, Inc. Cellules immunitaires déficientes en nr4a3 et leurs utilisations
WO2022256437A1 (fr) 2021-06-02 2022-12-08 Lyell Immunopharma, Inc. Cellules immunitaires déficientes en nr4a3 et leurs utilisations
WO2023064924A1 (fr) 2021-10-14 2023-04-20 Codiak Biosciences, Inc. Cellules productrices modifiées pour la production de vésicules extracellulaires
WO2023129974A1 (fr) 2021-12-29 2023-07-06 Bristol-Myers Squibb Company Génération de lignées de cellules avec site d'intégration
WO2023220603A1 (fr) 2022-05-09 2023-11-16 Regeneron Pharmaceuticals, Inc. Vecteurs et procédés de production d'anticorps in vivo
WO2023225665A1 (fr) 2022-05-19 2023-11-23 Lyell Immunopharma, Inc. Polynucléotides ciblant nr4a3 et leurs utilisations
WO2024064958A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a
WO2024064952A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a surexprimant c-jun

Also Published As

Publication number Publication date
CN101198694A (zh) 2008-06-11
CN101198694B (zh) 2013-04-10

Similar Documents

Publication Publication Date Title
WO2006097784A1 (fr) Variants de meganuclease i-crei presentant une specificite modifiee, leur procede de preparation, et leurs utilisations
AU2006224248B2 (en) I-Crei meganuclease variants with modified specificity, method of preparation and uses thereof
AU2007347328B2 (en) LAGLIDADG homing endonuclease variants having novel substrate specificity and use thereof
AU2006314220B2 (en) Laglidadg homing endonuclease variants having mutations in two functional subdomains and use thereof.
US20130061341A1 (en) Meganuclease variants cleaving a dna target sequence from a xp gene and uses thereof
WO2007060495A1 (fr) Variants de l'endonuclease homing i-crei a nouvelle specificite de clivage et leur utilisation
WO2008059382A2 (fr) Variants de méganucléase clivant une séquence cible d'adn à partir du gène hprt et leurs utilisations
CA2689305A1 (fr) Procede permettant d'ameliorer l'activite de clivage de meganucleases issues de i-crei

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

WWW Wipo information: withdrawn in national office

Country of ref document: RU

122 Ep: pct application non-entry in european phase

Ref document number: 05718441

Country of ref document: EP

Kind code of ref document: A1

WWW Wipo information: withdrawn in national office

Ref document number: 5718441

Country of ref document: EP