Granted Patent B2

US 12,577,583 · App. 18/611,182 · Granted Mar 17, 2026

Inventors: Bruce A. Hay (Encino, CA); Georg Oberhofer (Pasadena, CA); Tobin William Ivy (Pasadena, CA)

Assignee: California Institute of Technology

C12N15/85C12N9/22C12N15/907C12N2310/20C12N2800/40C12N2800/50C12N2810/10

Abstract

Claims (30)

1 . A composition comprising two vectors:

a first vector comprising:

a first sequence encoding a first component of a DNA sequence modifying complex, wherein the first component of the DNA sequence modifying complex is a nuclease, wherein the nuclease is Cas9 nuclease;

a second sequence encoding a second component of the DNA sequence modifying complex, wherein the second component of the DNA sequence modifying complex is a guide RNA, wherein the guide RNA enables the Cas9 nuclease to target specific sequences within the essential gene;

a first promoter operably linked to the first sequence encoding the first component of the DNA sequence modifying complex, wherein the first promoter comprises at least one of a germline promoter, a male specific germline promoter, a female specific germline promoter, a cell-type specific promoter, a tissue-specific promoter, a ubiquitous promoter, a promoter activated at a specific stage of mitosis, a promoter activated at a specific stage of meiosis, a viral promoter or prokaryotic promoter; and

a second promoter operably linked to the second sequence encoding the second component of the DNA sequence modifying complex, wherein the second promoter comprises at least one of a germline promoter, a male specific germline promoter, a female specific germline promoter, a cell-type specific promoter, a tissue-specific promoter, a ubiquitous promoter, a promoter activated at a specific stage of mitosis, a promoter activated at a specific stage of meiosis, a viral promoter or prokaryotic promoter;

wherein the DNA sequence modifying complex is configured to induce one or more sequence modifications in an endogenous copy of an essential gene;

a second vector comprising:

a rescue transgene sequence, wherein the rescue transgene is either a recoded copy of the essential gene or is a gene of unrelated sequence, wherein the rescue transgene encodes a protein that is functionally equivalent to a protein encoded by the essential gene; and

a rescue transgene promoter operably linked to the rescue transgene sequence, wherein the rescue transgene promoter comprises at least one of a endogenous promoter for the essential gene, germline promoter, a male specific germline promoter, a female specific germline promoter, a cell-type specific promoter, a tissue-specific promoter, a ubiquitous promoter, a promoter activated at a specific stage of mitosis, a promoter activated at a specific stage of meiosis, a viral promoter or prokaryotic promoter,

wherein the composition comprising two vectors is configured for reversibly modifying a population of organisms.

2 . The composition of claim 1 , wherein the nuclease cleaves and generates one or more double strand breaks in the endogenous copy of the essential gene.

3 . The composition of claim 2 , wherein the one or more double strand breaks are repaired to create an altered sequence of the essential gene.

4 . The composition of claim 1 , wherein the DNA sequence modifying complex does not modify the rescue transgene.

5 . The composition of claim 1 , wherein the composition comprising two vectors further comprises one or more cargo sequences.

6 . The composition of claim 5 , wherein the one or more cargo sequences comprise a one or more foreign gene sequences, or one or more alleles of an endogenous chromosomal or extra-chromosomal gene to which one of the vectors has been linked through nearby, or internal to the gene, insertion on the chromosome or extra-chromosomal element that carries the endogenous allele of interest.

7 . A method of reversibly modifying a population, the method comprising:

obtaining a wild type organism,

positioning the two vectors of claim 1 in the wild type organism

generating an altered organism by inducing one or more sequence modifications in an essential gene by a DNA sequence modifying complex that result in a defect in survival, growth control, fertility, or differentiation in one or more cells in the organism, and rescuing the defect in survival, growth control, fertility, or differentiation by a rescue transgene in the composition comprising two vectors,

introducing the altered organism in an environment wherein an increase in a frequency of the altered organism is desired relative to a frequency of the wild type organism in a population;

replacing the wild type organism with the altered organism in the population in the environment,

thereby obtaining a modified population

reintroducing the wild type organism in an environment wherein an increase in the frequency of the wild type organism is desired relative to the frequency of the altered organism in the modified population;

replacing the altered organism with the wild type organism in the modified population in the environment,

thereby reversibly modifying the population.

8 . The method of claim 7 , wherein the one or more cells comprise somatic cells, germline cells, gametes, or a combination thereof.

9 . The method of claim 7 , wherein the reversible modification of the population occurs at a rapid rate, high frequency, or both.

10 . The method of claim 9 , wherein the rapid rate is defined as replacement of at least 90% of the wild type organism by the altered organism or vice versa in the population after at most 100 generations.

11 . The method of claim 9 , wherein the high frequency is defined as replacement of at least 90% of the wild type organism by the altered organism or vice versa after 100 generations in the population.

Assignments (1)

Continuity (3)

References Cited (400)