# US 12,213,468 B2 · Population control using engineered translocations

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- Number: US 12,213,468 B2
- Document ID: US12213468B2
- Status: Granted
- Kind: B2
- Application number: 17/004,698
- Filed: 2020-08-27
- Published: 2020-12-31
- Granted: 2025-02-04
- Earliest claimed: 2015-05-26
- Assignees: California Institute of Technology
- Applicants: California Institute of Technology
- Inventors: Bruce A. Hay; Omar S. Akbari; Anna B. Buchman
- Web page: https://patentgrep.com/patent/US12213468B2

## CPC

- A01K 67/68: Genetically modified insects
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 2217/15: Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
- A01K 2227/706: Insects, e.g. Drosophila melanogaster, medfly
- A01K 2267/02: Animal zootechnically ameliorated
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Embodiments provided herein relate to systems for synthetically-engineered reciprocal chromosomal translocation for gene insertion into a population of organisms such as insects.

## Claims

### Claim 1

A method of distributing one or more genes of interest into a population of insects, the method comprising:
i) providing an insect population, wherein one or more individuals in the insect population comprises a translocation mediated gene drive system; and
ii) inducing a chromosomal translocation in the one or more individuals in the insect population,
wherein the chromosomal translocation generates a translocation-bearing altered insect population, wherein the translocation-bearing altered insect population comprises translocation-bearing individuals that are translocation heterozygotes and translocation-bearing individuals that are translocation homozygotes for the chromosomal translocation and wherein the translocation mediated gene drive system comprises:
a first construct, configured to be positionable at a first insertion site in a first chromosome, wherein the first construct comprises:
a) a first location to insert a first gene of interest;
b) a first promoter;
c) a first fragment of foreign stuffer DNA;
d) a second fragment of foreign stuffer DNA;
e) a first target site of a pair of target sites for an endonuclease positioned between the first and second fragments of foreign stuffer DNA;
f) a first splice acceptor site, positioned downstream from a-e; and
g) a first splice donor site, positioned between b and c, and a second construct, configured to be positionable at a second insertion site in a second chromosome, wherein the second construct comprises:
h) a second location to insert a second gene of interest;
i) a second promoter;
j) a third fragment of foreign stuffer DNA, wherein the third fragment is homologous to the second fragment;
k) a fourth fragment of foreign stuffer DNA, wherein the fourth fragment is homologous to the first fragment;
l) a second target site of the pair of target sites for an endonuclease positioned between the third and fourth fragments of foreign stuffer DNA;
m) a second splice acceptor site, positioned downstream from h-1, and
n) a second splice donor site, positioned between i and j, wherein the first and second chromosomes are non-homologous chromosomes,
wherein the first fragment of foreign stuffer DNA is homologous to the fourth fragment of foreign stuffer DNA and the second fragment of foreign stuffer DNA is homologous to the third fragment of foreign stuffer DNA,
wherein a double stranded break created at the first, and second target sites of the pair of target sites allows for homologous recombination between the first and fourth fragments, and between the second and third fragments upon a repair of the double stranded break (DSB),
wherein the repair of the DSB induces a chromosomal translocation and generates a first translocation chromosome and a second translocation chromosome.

### Claim 2 (depends on 1)

The method of claim 1, wherein one or more genes of interest in the translocation-bearing altered insect population encodes for a disease prevention protein/disease refractory protein, or a gene conferring conditional lethality, inability to undergo diapause, sterility, inability to fly.

### Claim 3 (depends on 1)

The method of claim 1, wherein the high frequency is defined as when translocation-bearing versions of chromosomes make up 90% or greater of the total chromosomes in a population as compared to wild type versions of the chromosomes involved in generating the translocation.

### Claim 4 (depends on 3)

The method of claim 3, wherein the rapid rate is defined as replacement of at least 90% of the wild type population chromosomes by the translocation-bearing altered insect population (translocation) after at most 5 generations.

### Claim 5 (depends on 1)

The method of claim 1, wherein the translocation-bearing altered insect population replaces 90% of the target wild type population after 30 generations.

### Claim 6 (depends on 1)

The method of claim 1, wherein the insect is a mosquito.

### Claim 7 (depends on 1)

The method of claim 1, wherein the condition in which translocation-based drive occurs comprises one or more of a population cage, a field cage, or an open environment.

### Claim 8 (depends on 1)

The method of claim 1, wherein the chromosomal translocation spreads to a high frequency within the wild population.

### Claim 9 (depends on 1)

The method of claim 1, wherein the translocation-bearing altered insect population is capable of replacing the wild type population at a rapid rate.

### Claim 10 (depends on 1)

The method of claim 1, wherein the insect is a psyllid.

### Claim 11

A population of translocation bearing insects comprising a gene drive system comprising:
a first construct, configured to be positionable at a first insertion site in a first chromosome, wherein the first construct comprises:
a) a first location to insert a first gene of interest;
b) a first promoter;
c) a first fragment of foreign stuffer DNA;
d) a second fragment of foreign stuffer DNA;
e) a first target site of a pair of target sites for an endonuclease positioned between the first and second fragments of foreign stuffer DNA;
f) a first splice acceptor site, positioned downstream from a-e; and
g) a first splice donor site, positioned between b and c, and a second construct, configured to be positionable at a second insertion site in a second chromosome, wherein the second construct comprises:
h) a second location to insert a second gene of interest;
i) a second promoter;
j) a third fragment of foreign stuffer DNA, wherein the third fragment is homologous to the second fragment;
k) a fourth fragment of foreign stuffer DNA, wherein the fourth fragment is homologous to the first fragment;
l) a second target site of the pair of target sites for an endonuclease positioned between the third and fourth fragments of foreign stuffer DNA;
m) a second splice acceptor site, positioned downstream from h-1, and
n) a second splice donor site, positioned between i and j, wherein the first and second chromosomes are non-homologous chromosomes,
wherein the first fragment of foreign stuffer DNA is homologous to the fourth fragment of foreign stuffer DNA and the second fragment of foreign stuffer DNA is homologous to the third fragment of foreign stuffer DNA,
wherein a double stranded break created at the first, and second target sites of the pair of target sites allows for homologous recombination between the first and fourth fragments, and between the second and third fragments upon a repair of the double stranded break (DSB),
wherein the repair of the DSB induces a chromosomal translocation and generates a first translocation chromosome and a second translocation chromosome.

### Claim 12 (depends on 11)

The population of translocation bearing insects of claim 11, wherein the insects are mosquitos.

### Claim 13 (depends on 11)

The population of translocation bearing insects of claim 11, wherein the insects are psyllids.

### Claim 14 (depends on 1)

The method of claim 1, wherein the translocation-bearing individuals display a fitness that is greater than that of wildtype individuals when challenged in a condition in which a translocation-based drive occurs,
wherein the fitness is defined as a population genetic behavior, over multiple generations, of translocation chromosomes in populations comprising both translocation-bearing individuals and non-translocation-bearing individuals,
wherein the chromosomal translocation is defined as having a higher fitness than wildtype under conditions that result in translocation-bearing chromosomes increasing in frequency over multiple generations,
wherein the translocation is defined as having a lower fitness than wildtype under conditions that result in translocation-bearing chromosomes decreasing in frequency over multiple generations, and
wherein fitness is frequency dependent such that a higher frequency of a first genotype results in an increase in relative fitness of the first genotype with respect to alternative genotypes.