Lentiviral protein delivery system for RNA-guided genome editing
AssigneeThe United States of America, as represented by the Secretary, Department of Health and Human Services
InventorsNaoya Uchida, Juan J. Haro Mora, John F. Tisdale
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Abstract
The invention is directed to a system comprising a lentivirus vector particle which encodes at least one guide RNA sequence that is complementary to a first DNA sequence in a host cell genome, a Cas9 protein, and optionally a donor nucleic acid molecule comprising a second DNA sequence. The invention also is directed to a method of altering a DNA sequence in a host cell using such a system, where the host cell can be in a human and the altered DNA can be of the human β-globin gene. The invention also is directed to a fusion protein comprising a Cas9 protein and a cyclophilin A (CypA) protein. The invention also is directed to sequences of vectors that can be used in the system and method.
Claims20
A method of forming a system, the system comprising:
- (a) a lentivirus vector particle comprising a lentiviral genome which does not contain a Cas9 gene and encodes at least one guide RNA sequence that is complementary to a first DNA sequence in a host cell genome, wherein the first DNA sequence encodes a defective human (β-globin protein, and
- (b) a Cas9 protein within the lentivirus vector particle;
- the method comprising:
- (1) obtaining the Cas9 protein as a non-fusion protein, and
- (2) packaging the non-fusion Cas9 protein into the lentivirus vector particle.
A vector having the sequence of SEQ ID NO:
- 35.
A method of forming a system, the system comprising:
- (a) a lentivirus vector particle comprising a lentiviral genome which does not contain a Cas9 gene and encodes at least two guide RNA sequences that are each complementary to a first DNA sequence in a host cell genome, and
- (b) a Cas9 protein within the lentivirus vector particle;
- the method comprising:
- (1) obtaining the Cas9 protein as a non-fusion protein, and
- (2) packaging the non-fusion Cas9 protein into the lentivirus vector particle.
A system comprising:
- (a) a lentivirus vector particle comprising a lentiviral genome which encodes at least one guide RNA sequence that is complementary to a first DNA sequence in a host cell genome, and
- (b) a Cas9 protein associated with the lentivirus vector, wherein the Cas9 protein is provided as a fusion protein comprising a Cas9 protein and a cyclophilin A (CypA) protein, and wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:
- 2 or SEQ ID NO:
- 3.
Dependencies are inferred from the claim text.