Modified iscb nucleases, modified guide rnas, and uses thereof
AssigneeAera Therapeutics, Inc.
InventorsLiyang ZHANG, Max Atticus ENGLISH, Colin McGAW, Hannah KEMPTON, Sean BENLER, Ian SLAYMAKER
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Abstract
Disclosed herein are novel polypeptides having nuclease activity. The polypeptides can effect modifications at defined locations within target nucleic acids, e.g., DNA and RNA. Also disclosed herein modified guide RNAs. The polypeptides and guide RNAs are useful in compositions, systems and methods, to make site specific alterations of target nucleic acid sequences.
Claims352
An IscB protein (protein effector polypeptide) comprising:
- (a) an amino acid sequence that is at least 80% identical to the amino acid sequence of an IscB as provided by any one of SEQ ID NO:
- 2, wherein the amino acid sequence of the IscB protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations in an amino acid residue selected from the group consisting of amino acid residues 18, 34, 36, 37, 60, 66, 85, 97, 99, 100, 101, 104, 109, 112, 118, 136, 137, 155, 156, 157, 166, 168, 190, 204, 204, 205, 206, 208, 212, 214, 216, 218, 219, 229, 229, 230, 233, 233, 234, 234, 238, 239, 241, 242, 245, 246, 248, 252, 254, 255, 256, 257, 258, 279, 280, 291, 293, 298, 303, 304, 306, 307, 312, 319, 323, 328, 329, 331, 332, 338, 341, 353, 360, 368, 372, 376, 377, 378, 379, 380, 381, 382, 384, 385, 386, 388, 390, 391, 393, 405, 406, 408, 409, 410, 412, 413, 416, 417, 418, 419, 421, 421, 423, 425, 437, 439, 455, 462, 463, 478, 479, 533, 536, 564 and 565 of the amino acid sequence of SEQ ID NO:
- 2 (pEDIT071), and provided that, if the IscB comprises a single mutation, it is not Q60I, N66H, I85R, I85K, E137R, P405R, D413K, E409R or combination thereof;
- or
- (b) an amino acid sequence that is at least 80% identical to the amino acid sequence of an IscB as provided by any one of SEQ ID NO:
- 8, wherein the amino acid sequence of the IscB protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations in an amino acid residue selected from the group consisting of amino acid residues 30, 46, 48, 49, 72, 78, 97, 109, 111, 112, 113, 116, 121, 124, 130, 148, 149, 167, 168, 169, 178, 180, 202, 216, 216, 217, 218, 220, 224, 226, 228, 230, 231, 241, 241, 242, 245, 245, 246, 246, 250, 251, 253, 254, 257, 258, 260, 264, 266, 267, 268, 269, 270, 291, 292, 303, 305, 310, 315, 316, 318, 319, 324, 331, 335, 340, 341, 343, 344, 350, 353, 365, 372, 380, 384, 388, 389, 390, 391, 392, 393, 394, 396, 397, 398, 400, 402, 403, 405, 417, 418, 420, 421, 422, 424, 425, 428, 429, 430, 431, 433, 433, 435, 437, 449, 451, 467, 474, 475, 490, 491, 545, 548, 576, and 577 of the amino acid sequence of SEQ ID NO:
- 8 (pEDIT071+NLS), and provided that, if the IscB comprises a single mutation, and optionally, if the IscB comprises a single mutation, the mutation is not Q72I, H78H, I97R, 97K, E149R, P417R, E421R, D425K, or combination thereof.
A ωRNA comprising a conserved portion of a ωRNA (parent ωRNA) comprising one or more stem-loop structures, wherein at least one of the stem-loop structures comprises a modification, and wherein a conserved portion of the ωRNA is shorter than the conserved portion of the parent ωRNA and is capable of binding with a transposon family encoded nuclease or a homolog or ortholog thereof.
The kit of 274 or 275, further comprising an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone.
Dependencies are inferred from the claim text.