1. A single-chain I-CreI meganuclease comprising two domains in the orientation N-terminal toward C-terminal joined by a peptidic linker, wherein:
(a) each domain, derived from a parent I-CreI monomer, comprises a portion of said parent I-CreI monomer said portion extending at least from the beginning of the first alpha helix (\u03b11) to the end of the C-terminal loop of I-CreI and including successively: the \u03b11\u03b21\u03b22\u03b12\u03b23\u03b24\u03b13core domain, the \u03b14 and \u03b15 helices and the C-terminal loop of I-CreI, and
(b) the two domains are joined by a peptidic linker which allows said two domains to fold as a I-CreI dimer that is able to bind and cleave a chimeric DNA target comprising one different half of each parent homodimeric I-CreI meganuclease target sequence, wherein said peptidic linker is selected from the group consisting of the sequences SEQ ID NO: 2 to 12 and 14 to 19.
2. The single-chain I-CreI meganuclease according to claim 1, wherein
(i) the N-terminal domain starts at position 1, 2, 3, 4, 5, or 6 of I-CreI (SEQ ID NO:20) andor the C-terminal domain starts at position 2, 3, 4, 5, or 6 of I-CreI (SEQ ID NO:20); and
(ii) the N-terminal andor C-terminal domain(s) terminate(s) at position 145 of I-CreI (SEQ ID NO:20); or
(iii) the N-terminal andor C-terminal domain(s) further include(s) at least the \u03b16 helix of I-CreI (SEQ ID NO:20); or
(iv) the N-terminal andor C-terminal domain(s) terminate(s) at position 152, 156, 160 or 163 of I-CreI (SEQ ID NO:20).
3. The single-chain I-CreI meganuclease according to claim 1, wherein each domain comprises mutations at positions 26 to 40 andor 44 to 77 of I-CreI (SEQ ID NO:20) , said single-chain I-CreI meganuclease being able to cleave a non-palindromic DNA target sequence.
4. The single-chain I-CreI meganuclease according to claim 1, wherein at least one domain comprises a mutation at positions 137 to 143 of I-CreI (SEQ ID NO:20) that modifies the specificity of the single-chain I-CreI meganuclease towards the I-CreI site of SEQ ID NO:21.
5. The single-chain I-CreI meganuclease according to claim 1, which comprises one or more mutations that impair the formation of functional homodimers from the two domains.
6. The single-chain I-CreI meganuclease according to claim 5, wherein each domain comprises at least one mutation, selected from the group consisting of:
K7E or K7D and E8K or E8R; F54G or F54A and L97F or L97W; K96D or K96E and E61R or E611K; R51D or R51E and D137R or D137K of SEQ ID NO: 20, respectively for the first and the second domain.
7. The single-chain I-CreI meganuclease according to claim 6, wherein one domain comprises the substitution of the lysine residues at positions 7and 96 of SEQ ID NO: 20 by an acidic amino acid and the other domain comprises the substitution of the glutamic acid residues at positions 8and 61 of SEQ ID NO: 20 by a basic amino acid.
8. The single chain I-CreI meganuclease according to claim 1 wherein one domain comprises the G19S mutation of SEQ ID NO: 20.
9. The single-chain I-CreI meganuclease according to claim 1, which comprises the sequence SEQ ID NO: 95or 97.
10. A pharmaceutical composition comprising at least one single-chain I-CreI meganuclease of claim 1.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A carabiner comprising:
a generally C-shaped body comprising a first end and a second end located on opposite sides of an opening;
a primary gate mounted to the second end and rotatable about a first axis perpendicular to a plane that bisects the C-shaped body, the primary gate rotatable between a closed position with the primary gate engaged with the first end and the C-shaped body enclosing an interior region, and an open position with the primary gate located generally in the interior region; and
a secondary gate comprising a first end attached to the primary gate and a second free end having a portion adapted to engage an interior surface of the C-shaped body, the portion of the second free end constrained to move only in the interior region, the secondary gate adapted to be displaced in the plane relative to the primary gate between a closed configuration that divides the interior region into a first portion and a second portion, and an open configuration where the interior region is accessible through the opening, wherein the secondary gate is adapted to move between the open configuration and the closed configuration as the primary gate moves between the open position and the closed position.
2. The carabiner of claim 1 wherein the secondary gate is pivotally attached to the primary gate.
3. The carabiner of claim 2 wherein the portion of the secondary gate is biased to slide along the interior surface of the C-shaped body as the primary gate rotates between the closed position and the open position.
4. The carabiner of claim 2 wherein the secondary gate is biased toward the open configuration, the carabiner comprising a locking mechanism on the primary gate that simultaneously advances the secondary gate toward the closed configuration as the locking mechanism is advanced toward a locked position.
5. The carabiner of claim 1 wherein the secondary gate is adapted to move in the plane to extend across the interior region as the primary gate rotates between the open position and the closed position.
6. The carabiner of claim 1 wherein engagement of the second free end of the secondary gate with the interior surface of the C-shape body is sufficient to prevent the carabiner from rotating in a belay loop.
7. The carabiner of claim 1 wherein engagement of the second free end of the secondary gate with the interior surface of the C-shape body is sufficient to prevent a rope from moving from the first portion to the second portion.
8. The carabiner of claim 1 comprising a locking mechanism that engages with the primary gate and retains the secondary gate in the closed configuration.
9. The carabiner of claim 1, wherein the second distal end of the secondary gate rotates is toward the second end of the C-shaped body as the primary gate rotates toward the open position.
10. The carabiner of claim 1 wherein engagement of the second free end of the secondary gate with the interior surface of the C-shape body is sufficient to prevent cross-loading of the carabiner during use.
11. The carabiner of claim 1 wherein the secondary gate is attached proximate a distal end of the primary gate.
12. A carabiner comprising:
a generally C-shaped body comprising a first end and a second end at opposite sides of an opening;
a primary gate mounted to the second end and rotatable about a first axis perpendicular to a plane that bisects the C-shaped body, the primary gate rotatable between a closed position with the primary gate engaged with the first end and the C-shaped body enclosing an interior region, and an open position with the primary gate located generally in the interior region; and
a secondary gate comprising a first end attached to the primary gate and a second free end of the secondary gate constrained to move only in the interior region, the secondary gate adapted to be displaced in the plane relative to the primary gate between a closed configuration that divides the interior region into a first portion and a second portion, and an open configuration where the interior region is accessible through the opening, wherein the secondary gate is adapted to move in the plane to extend across the interior region as the primary gate rotates between the open position and the closed position.
13. The carabiner of claim 12 wherein the secondary gate is adapted to move between the open and closed configurations as the primary gate moves between the open and closed positions.
14. The carabiner of claim 12 wherein the secondary gate is adapted to prevent the carabiner from rotating in a belay loop.
15. The carabiner of claim 12 wherein the secondary gate is adapted to prevent cross-loading of the carabiner.
16. The carabiner of claim 12 wherein the secondary gate is pivotally attached to the primary gate.
17. The carabiner of claim 12 wherein the secondary gate is adapted to prevent a rope from moving from the first portion to the second portion.
18. A method of making a carabiner comprising the steps of:
mounting a primary gate to a second end of a generally C-shaped body for rotation about a first axis perpendicular to a plane that bisects the C-shaped body, the primary gate rotatable between a closed position with the primary gate engaged with a first end of the C-shaped body and an open position with the primary gate located in an interior region of the C-shaped body;
attaching a first end of a secondary gate to the primary gate so that a second free end of the secondary gate is constrained to move only in the interior region in the plane of the primary gate; and
rotating the primary gate between the closed position and the open position to rotate the secondary gate in the plane between a closed configuration where the secondary gate extends across the interior region to divide the interior region into a first portion and a second portion, and an open configuration where the interior region is accessible through the opening.