Mutant cytidine deaminases with improved editing precision
AssigneeCorrectsequence Therapeutics
InventorsLijie WANG, Tao WEI, Yichuan WANG, Xiaodun MOU
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Abstract
Provided are mutant cytidine deaminases and related molecules useful for conducting base editing with reduced or no off-target mutations and with improved editing site precision. The mutant catalytic domain of the mouse APOBEC3 protein includes one or more mutations which helps to narrow the editing window while maintaining high editing efficiency. Example mutations include Y35D and K40H-W102Y. Also provided are improved base editing systems and methods using these mutant cytidine deaminases.
Claims25
A protein, comprising a catalytic domain of a mutant mouse APOBEC3 protein, wherein the catalytic domain has at least 85% sequence identity to amino acid residues 35-141 of SEQ ID NO:
- 1 and comprises a substitution, relative to SEQ ID NO:
- 1, at a residue selected from the group consisting of Y35, K37, R39, K40, N66, W102, Y132, and combinations thereof.
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A polynucleotide, comprising
- a first fragment encoding (a) a first fusion protein comprising a nucleobase deaminase, a nucleobase deaminase inhibitor, and a first RNA recognition peptide, wherein the nucleobase deaminase and the nucleobase deaminase inhibitor is separated by a protease cleavage site that can be cleaved by a protease;
- a second fragment encoding (b) a second fusion protein comprising an inactive portion of the protease fused to a second RNA recognition peptide that is different from the first RNA recognition peptide;
- a third fragment encoding (c) a second portion of the protease which, in combination with the first portion, can carry out the protease activity to cleave the protease cleavage site;
- a fourth fragment encoding (d) a helper sgRNA further comprising a first RNA recognition site recognizable by first RNA recognition peptide;
- and
- a fifth fragment encoding (e) a sgRNA further comprising a second RNA recognition site recognizable by the second RNA recognition peptide,
- wherein the first, second, and third fragments are configured to be transcribed into a single mRNA molecule, wherein
- (1) the first and second fragments are separated by a first separating sequence encoding a first internal ribosome entry site (IRES), and the second and third fragments are separated by a second separating sequence encoding a first self-cleavage peptide;
- or
- (2) the first and second fragments are separated by a first separating sequence encoding a second self-cleavage peptide, and the second and third fragments are separated by a second separating sequence encoding a second internal ribosome entry site (IRES).
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Dependencies are inferred from the claim text.