METHODS AND KITS FOR DYNAMIC TARGETED HYPERMUTATION
First Claim
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1. A nucleobase-editing fusion protein comprising a processive polynucleic acid-binding protein fused to a nucleobase-editing enzyme which is capable of altering nucleobases in a pre-existing polynucleic acid sequence.
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Abstract
Disclosed herein are methodologies and kits for dynamic targeted hypermutation that harness the enzymatic activity of a polynucleic acid-binding protein fused to a nucleobase-editing enzyme to specifically target mutations across a region of interest. These methodologies and kits facilitate the rapid creation of diverse DNA libraries in vivo or in vitro.
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37 Claims
- 1. A nucleobase-editing fusion protein comprising a processive polynucleic acid-binding protein fused to a nucleobase-editing enzyme which is capable of altering nucleobases in a pre-existing polynucleic acid sequence.
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9. A method of performing dynamic targeted hypermutation comprising contacting at least one polynucleic acid with at least one non-naturally occurring nucleobase-editing fusion protein, wherein:
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a. each of the at least one non-naturally occurring nucleobase-editing fusion proteins comprises a processive polynucleic acid-binding protein fused to a nucleobase-editing enzyme; b. each of the at least one polynucleic acid comprises a target region; and c. the contacting of the at least one polynucleic acid with the at least one non-naturally occurring nucleobase-editing fusion protein generates mutations at a rate exceeding background mutation rates only in the target region of the at least one polynucleic acid of (b), wherein the background mutation rate of the at least one polynucleic acid of (b) is determined in the absence of the non-naturally occurring nucleobase-editing fusion protein. - View Dependent Claims (10, 11, 13, 17, 18, 21, 23, 24, 26)
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27. A kit for performing dynamic targeted hypermutation comprising:
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a. a polypeptide comprising the amino acid sequence of a non-naturally occurring nucleobase-editing fusion protein comprising a processive polynucleic acid-binding protein fused to a nucleobase-editing enzyme or a polynucleic acid sequence encoding for and driving the expression of said polypeptide; and b. a polynucleic acid sequence comprising, from 5′
to 3′
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a promoter region that is bound by the non-naturally occurring nucleobase-editing fusion protein of (a) in a sequence-specific manner;
a cloning site; and
a terminator region comprising a terminator array. - View Dependent Claims (29, 30, 32, 36)
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Specification