System for genome editing
AssigneesPresident and Fellows of Harvard College, The Broad Institute, Inc.
InventorsDavid R. Liu, James William Nelson
Life of this patent
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Abstract
The present specification provides compositions and methods that are capable of directly installing an insertion or deletion of a given nucleotide at a specified genetic locus. The compositions and methods involve the novel combination of the use an engineered RNA enzyme (i.e., “ribozyme”) that is capable of site-specifically inserting or deleting a single nucleotide at a genetic locus and the use of a nucleic acid programmable DNA binding protein (napDNAbp) (e.g., Cas9) to target the engineered ribozyme to a specified genetic locus, thereby allowing for the direct installation of an insertion of deletion at the specified genetic locus by the engineered ribozyme.
Claims106
An engineered ribozyme represented by the structure of FIG. 1A.
An engineered ribozyme represented by the structure of FIG. 3B.
An engineered ribozyme comprising a deletion in the 3′ terminal end sufficient to remove the self-insertion activity of the ribozyme.
A ribozyme-mediated programmable nucleic acid editing construct comprising a ribozyme and a nucleic acid programmable DNA binding protein (napDNAbp) which is capable of installing an insertion of one or more nucleotides at a target site in a DNA molecule.
A method for introducing a new nucleobase pair into a target site of a DNA molecule, comprising contacting a single-stranded R-loop formed in the DNA molecule by a bound napDNAbp with an engineered ribozyme, wherein the engineered ribozyme is configured to insert a nucleobase into an insertion site located in the R-loop.
An engineered ribozyme comprising SEQ ID NO:
- 88, or a ribozyme comprising a nucleotide sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO:
- 88.
An engineered ribozyme comprising SEQ ID NO:
- 89, or a ribozyme comprising a nucleotide sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO:
- 89.
An engineered ribozyme comprising SEQ ID NO:
- 156, or a ribozyme comprising a nucleotide sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO:
- 156.
An engineered ribozyme comprising SEQ ID NO:
- 157, or a ribozyme comprising a nucleotide sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO:
- 157.
A genome editing system comprising a nucleic acid programmable DNA binding protein (napDNAbp), a guide RNA, and a ribozyme.
An engineered ribozyme that catalyzes the insertion of a nucleotide into a single-stranded DNA molecule.
Dependencies are inferred from the claim text.