RNA-Guided Gene Drives

Patent metadata

Field	Value
Publication number	US-2016333376-A1
Application number	US-201615172645-A
Country	US
Kind code	A1
Filing date	Jun 3, 2016
Priority date	Jan 8, 2014
Publication date	Nov 17, 2016
Grant date	—

Abstract

RNA guided Cas9 gene drives and method for their use are disclosed.

First claim

1 . A method of altering a eukaryotic germline cell of an organism comprising
introducing into the germline cell a first foreign nucleic acid sequence encoding an RNA guided DNA binding protein nuclease and one or more guide RNAs, and including corresponding promoter sequences and a first flanking sequence and a second flanking sequence, wherein the one or more guide RNAs are complementary to one or more target locations on genomic DNA of a first chromosome and a second chromosome of a chromosome pair of the germline cell, wherein the nucleic acid sequence encoding the RNA guided DNA binding protein nuclease and the nucleic acid sequence encoding the one or more guide RNAs are between the first flanking sequence and the second flanking sequence, wherein the first flanking sequence includes a first sequence identical to a first portion of the target location on the first chromosome or the second chromosome of the genomic DNA, wherein the second flanking sequence includes a second sequence identical to a second portion of the target location on the first chromosome or the second chromosome of the genomic DNA, expressing the first foreign nucleic acid sequence to produce the RNA guided DNA binding protein nuclease and the one or more RNAs wherein the RNA guided DNA binding protein nuclease and an associated guide RNA co-localize to an associated target location on the first chromosome of the genomic DNA and the second chromosome of the genomic DNA and the RNA guided DNA binding protein nuclease cleaves the first chromosome of the genomic DNA at the target location in a cleavage site specific manner and cleaves the second chromosome of the genomic DNA at the target location in a cleavage site specific manner, inserting the first foreign nucleic acid sequence into the first chromosome of the chromosome pair of the genomic DNA at the cleavage site, and inserting the first foreign nucleic acid sequence into the second chromosome of the chromosome pair of the genomic DNA at the cleavage site to render the germline cell homozygous for the foreign nucleic acid sequence, and further comprising introducing into the germline cell a second foreign nucleic acid sequence encoding an RNA guided DNA binding protein nuclease and one or more guide RNAs, and including corresponding promoter sequences and a first flanking sequence and a second flanking sequence, wherein the one or more guide RNAs are complementary to the one or more target locations that were altered by the first foreign nucleic acid sequence, expressing the second foreign nucleic acid sequence to produce the RNA guided DNA binding protein nuclease and the one or more RNAs wherein the RNA guided DNA binding protein nuclease and an associated guide RNA co-localize to an associated target location on the chromosome of the genomic DNA and the RNA guided DNA binding protein nuclease cleaves the chromosome of the genomic DNA at the target location in a cleavage site specific manner to remove the first foreign nucleic acid sequence, and inserting the second foreign nucleic acid sequence into the chromosome of the genomic DNA at the cleavage site.

2 . The method of claim 1 wherein the RNA guided DNA binding protein nuclease creates a double stranded break or a single stranded break.

3 . The method of claim 1 wherein the RNA guided DNA binding protein nuclease is Cas9.

4 . (canceled)

5 . The method of claim 1 wherein the RNA guided DNA binding protein nuclease is Cas9 nickase.

6 . The method of claim 1 wherein at least one target location is within an essential gene and the first foreign nucleic acid sequence is inserted adjacent to and replacing part of the essential gene.

7 . The method of claim 1 wherein the first foreign nucleic acid sequence is inserted by homologous recombination.

8 . The method of claim 1 wherein the germline cell is a fungal cell, a plant cell, an insect cell or a mammalian cell.

9 . The method of claim 1 wherein the one or more guide RNAs includes between about 10 to about 250 nucleotides.

10 . (canceled)

11 . The method of claim 1 wherein the RNA guided DNA binding protein nuclease co-localizes with an associated guide RNA at a plurality of associated target locations to cleave either the first chromosome or the second chromosome at a plurality of cleavage sites.

12 . The method of claim 11 wherein cleaving at the plurality of cleavage sites removes a DNA sequence which is replaced with the first foreign nucleic acid sequence by homologous recombination.

13 .- 22 . (canceled)

23 . The method of claim 1 wherein the second foreign nucleic acid sequence includes genetic modifications different from the first foreign nucleic acid sequence.

24 . The method of claim 1 wherein the second foreign nucleic acid sequence includes the wild type sequence of the chromosome that was modified by the first foreign nucleic acid sequence.

25 .- 29 . (canceled)

30 . A method of altering a eukaryotic germline cell of an organism at a plurality of genes comprising
introducing into the germline cell a first foreign nucleic acid sequence encoding an RNA guided DNA binding protein nuclease and a plurality of guide RNAs, and including corresponding promoter sequences and a first flanking sequence and a second flanking sequence, along with one or more additional foreign nucleic acid sequences corresponding to the plurality of genes, wherein the plurality of guide RNAs include one or more gene specific guide RNAs that are complementary to one or more target locations on a plurality of genes in the genomic DNA of a chromosome of the germline cell, wherein the nucleic acid sequence encoding the RNA guided DNA binding protein nuclease and the nucleic acid sequence encoding the plurality of guide RNAs are between the first flanking sequence and the second flanking sequence, wherein the first flanking sequence includes a first sequence identical to a first portion of one of the target locations on the chromosome of the genomic DNA, wherein the second flanking sequence includes a second sequence identical to a second portion of the same target location on the chromosome of the genomic DNA, wherein each of the one or more nucleic acid sequences contain a first flanking sequence and a second flanking sequence corresponding to a first portion and a second portion of the remaining target locations, expressing the first foreign nucleic acid sequence to produce the RNA guided DNA binding protein nuclease and the plurality of RNAs wherein the RNA guided DNA binding protein nuclease and associated guide RNAs co-localize to a plurality of associated target locations on the chromosome of the genomic DNA and the RNA guided DNA binding protein nuclease cleaves the first chromosome of the genomic DNA at a plurality of genes in a cleavage site specific manner, inserting the first foreign nucleic acid sequence into the chromosome of the genomic DNA at the cleavage site, and further comprising introducing into the germline cell a second foreign nucleic acid sequence encoding an RNA guided DNA binding protein nuclease and a plurality of guide RNAs, and including corresponding promoter sequences and a first flanking sequence and a second flanking sequence, wherein the plurality of guide RNAs are complementary to the plurality of target locations that were altered by the first foreign nucleic acid sequence, expressing the second foreign nucleic acid sequence to produce the RNA guided DNA binding protein nuclease and the plurality of guide RNAs wherein the RNA guided DNA binding protein nuclease and associated guide RNAs co-loc

Assignees

• Harvard College

Inventors

• Esvelt Kevin M
• Smidler Andrea L

Classifications

• C12N15/902

  using homologous recombination · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/905 Primary

  in yeast · CPC title