Engineered CAS9 systems for eukaryotic genome modification
AssigneeSigma-Aldrich Co.
InventorsTimothy Seebeck, Fuqiang Chen, Gregory D. Davis
Life of this patent
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Abstract
Engineered Cas9 systems that utilize alternate protospacer adjacent motifs for target DNA binding, nucleic acids encoding the engineered Cas9 systems, and methods of using the engineered Cas9 systems for modifying target chromosomal sequences in eukaryotic cells.
Claims26
A system for eukaryotic genome modification comprising (a) an engineered Bacillus smithii Cas9 protein comprising a nuclear localization signal (NLS), and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Bacillus smithii Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T.
A plurality of nucleic acids encoding a system for eukaryotic genome modification comprising (a) an engineered Bacillus smithii Cas9 protein comprising a nuclear localization signal (NLS);
- and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Bacillus smithii Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T;
- the plurality of nucleic acids comprising at least one nucleic acid encoding the engineered Bacillus smithii Cas9 protein, and at least one nucleic acid encoding the engineered guide RNA.
A eukaryotic cell comprising at least one system comprising (a) an engineered Bacillus smithii Cas9 protein comprising a nuclear localization signal (NLS);
- and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Bacillus smithii Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T, wherein the eukaryotic cell is a non-human mammalian cell, a plant cell, a non-mammalian vertebrate cell, an invertebrate cell, or a single cell eukaryotic organism and wherein the eukaryotic cell is in vivo, ex vivo, or in vitro.
A human eukaryotic cell comprising at least one system comprising (a) an engineered Bacillus smithii Cas9 protein comprising a nuclear localization signal (NLS);
- and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Bacillus smithii Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NNNNCAAA-3′, wherein the human eukaryotic cell is ex vivo, or in vitro.
A fusion protein comprising an engineered Bacillus smithii Cas9 protein linked to at least one chromatin modulating motif,
- wherein the chromatin modulating motif is a high mobility group (HMG) box (HMGB) DNA binding domain, a HMG nucleosome-binding (HMGN) protein, a central globular domain from a histone H1 variant comprising SEQ ID NO:
- 74, or a combination thereof,
- wherein the at least one chromatin modulating motif is linked to the Cas9 protein at its N-terminus, C-terminus, or a combination thereof,
- wherein, together with an engineered guide RNA designed to complex with the Cas9 protein, the fusion protein is capable of hybridizing with a target sequence in a double-stranded sequence, and
- wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T.
Dependencies are inferred from the claim text.