Genetic data compression and methods of use

Patent metadata

Field	Value
Publication number	US-2021332387-A1
Application number	US-202117236556-A
Country	US
Kind code	A1
Filing date	Apr 21, 2021
Priority date	Apr 21, 2020
Publication date	Oct 28, 2021
Grant date	—

Abstract

Provided herein are genetically modified cells and methods of their production, wherein such methods include introducing a nucleic acid molecule including a plurality of index sequences into a cell comprising a synthetic landing pad, wherein each of the plurality of index sequences includes a first portion of a sequence and the synthetic landing pad includes a second portion of the sequence. The method further includes generating a plurality of cells that include the synthetic landing pad and the nucleic acid molecule including the plurality of index sequences and integrating one of the plurality of index sequences into the synthetic landing pad in each of the cells, thereby linking the first and second portions of the sequence. The linked first and second portions of the sequence result in a functional gene and cells including the integrated index sequence are selected based on presence or activity of the functional gene.

First claim

We claim:

1 . A method of producing a plurality of genetically modified cells, comprising:
introducing a nucleic acid molecule comprising a plurality of index sequences into a cell comprising a genomic polynucleotide comprising a synthetic landing pad, wherein each of the plurality of index sequences comprises a first portion of a nucleotide sequence and the synthetic landing pad comprises a second portion of the nucleotide sequence to produce a cell comprising the synthetic landing pad and the nucleic acid molecule comprising the plurality of index sequences; generating a plurality of progeny cells comprising the genomic polynucleotide comprising the synthetic landing pad and the nucleic acid molecule comprising the plurality of index sequences; integrating a single index sequence into the synthetic landing pad in each of the plurality of progeny cells, thereby linking the first and second portions of the nucleotide sequence; and selecting progeny cells comprising integrated index sequences based on presence or activity of the linked first and second portions of the nucleotide sequence, thereby producing a plurality of genetically modified cells.

2 . The method of claim 1 , wherein the nucleic acid molecule comprising the plurality of index sequences comprises 500-3,000 index sequences.

3 . The method of claim 1 , wherein the first portion and the second portion of the nucleotide sequence reconstitute a functional gene when linked.

4 . The method of claim 3 , wherein the functional gene is a selectable marker or reporter gene.

5 . The method of claim 3 , wherein the synthetic landing pad further comprises a site-specific nuclease (SSN) recognition site and homology arms flanking the SSN recognition site, and each of the plurality of index sequences is flanked by the homology arms in the nucleic acid molecule comprising the plurality of index sequences.

6 . The method of claim 5 , wherein each of the homology arms is 150-500 nucleotides in length.

7 . The method of claim 5 , wherein integrating the single index sequence into the synthetic landing pad comprises introducing a DNA break at the SSN recognition site utilizing the SSN, and site-specific integration of the index sequence into the synthetic landing pad.

8 . The method of claim 7 , wherein the SSN is a Cas, zinc-finger nuclease, or TALEN.

9 . The method of claim 1 , wherein the nucleic acid molecule comprising the plurality of index sequences is an extrachromosomal array, a plasmid, or an artificial chromosome.

10 . The method of claim 1 , wherein each of the plurality of index sequences comprises a homologous fragment of the genomic polynucleotide, and wherein each of the plurality of index sequences are different.

11 . The method of claim 10 , wherein the genomic polynucleotide is an intron or exon of a gene, or a promoter element.

12 . The method of claim 1 , wherein the cell is a eukaryotic cell or bacterial cell.

13 . The method of claim 12 , wherein the cell is a yeast cell, mammalian cell, a Caenorhabditis elegans cell, or a Drosophila cell.

14 . The method of claim 12 , wherein the lineage of the cell is traced by detecting an index sequence in progeny of at least one of the plurality of genetically modified cells.

15 . The method of claim 1 , wherein introducing the nucleic acid molecule comprising the plurality of index sequences into the cell comprises injecting the nucleic acid molecule into an animal comprising the cell.

16 . The method of claim 1 , wherein each of the plurality of index sequences comprises a sequence variant of a reference coding sequence, a sequence variant of a reference non-coding sequence, a library sequence, a randomized sequence, or a promoter element.

17 . The method of claim 16 , further comprising:
selecting a single sequence variant of the reference coding sequence by selecting a genetically modified cell comprising the reference coding sequence variant; selecting a single sequence variant of the reference non-coding sequence by selecting a genetically modified cell comprising the reference non-coding sequence variant; selecting a single library sequence by selecting a genetically modified cell comprising the library sequence; selecting a single randomized sequence by selecting a genetically modified cell comprising the randomized sequence; or selecting a single promoter element by selecting a genetically modified cell comprising a screenable marker or reporter gene operably linked to the promoter element in the genomic polynucleotide.

18 . The method of claim 1 , further comprising:
selecting a genetically modified cell comprising an index sequence by an assay phenotype, or by expression of a selectable marker or reporter; generating variants of the index sequence; introducing a nucleic acid molecule comprising the variants of the index sequence into a cell comprising a genomic polynucleotide comprising a synthetic landing pad, wherein each of the variants of the index sequence comprises a first portion of a nucleotide sequence and the synthetic landing pad comprises a second portion of the nucleotide sequence to produce a cell comprising the synthetic landing pad and the nucleic acid molecule comprising the variants of the index sequence; generating a plurality of progeny cells comprising the genomic polynucleotide comprising the synthetic landing pad and the nucleic acid molecule comprising the variants of the index sequence; integrating a single variant of the index sequence into the synthetic landing pad in each of the plurality of progeny cells, thereby linking the first and second portions of the nucleotide sequence; and selecting progeny cells comprising integrated variants of the index sequence based on presence or activity of the linked first and second portions of the nucleotide sequence.

19 . A genetically modified cell comprising:
an extrachromosomal array comprising a plurality of index sequences; and a genomic polynucleotide comprising one of the plurality of index sequences integrated at a synthetic landing pad, wherein the integrated index sequence comprises a first portion of a nucleotide sequence and the synthetic landing pad comprises a second portion of the nucleotide sequence, and wherein the first and second portions of the nucleotide sequence are operably linked in the genomic polynucleotide.

20 . A multicellular organism comprising a plurality of the genetically modified cells of claim 19 , wherein the genetically modified cells comprise different index sequences.

Assignees

• Univ Oregon

Inventors

• Stevenson Zachary C
• Banse Stephen A
• Phillips Patrick C

Classifications

• A01K2227/703

  Worms, e.g. Caenorhabdities elegans · CPC title

• A01K67/64

  Genetically modified nematodes · CPC title

• A01K2267/0393

  Animal model comprising a reporter system for screening tests · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title

• C12Q1/6809

  Methods for determination or identification of nucleic acids involving differential detection · CPC title