Production and tracking of engineered cells with combinatorial genetic modifications

Patent metadata

Field	Value
Publication number	US-12577557-B2
Application number	US-202017429250-A
Country	US
Kind code	B2
Filing date	Feb 7, 2020
Priority date	Feb 8, 2019
Publication date	Mar 17, 2026
Grant date	Mar 17, 2026

Abstract

Described herein are methods for making genetically modified cells by introducing combinations of genetic variants (designed or random) or constructs (genes or otherwise arbitrary DNA) into a population of cells, and for tracking each variant combination by sequentially building an array of barcodes at a common locus (chromosomal or plasmid), termed the barcode locus. Also described are the cells made by such methods.

First claim

What is claimed is:

1 . A method for producing a plurality of genetically engineered cells, the method comprising:
(a-1) contacting a plurality of cells with a first gene editing agent and a first plurality of recombinant polynucleotides, each recombinant polynucleotide in the first plurality comprising a first unique polynucleotide barcode sequence, thereby forming a first plurality of gene edited and barcoded cells, each gene edited and barcoded cell comprising the first unique polynucleotide barcode inserted into a barcode locus, and a first unique genetic modification; and (b-1) contacting the first plurality of gene edited and barcoded cells with a second gene editing agent and a second plurality of recombinant polynucleotides, each recombinant polynucleotide in the second plurality comprising a second unique polynucleotide barcode sequence, thereby forming a second plurality of gene edited and barcoded cells, each gene edited and barcoded cell comprising the second unique polynucleotide barcode inserted into a barcode locus and a second unique genetic modification, or (a-2) contacting a plurality of cells with a first gene editing agent thereby producing a first plurality of gene edited cells each comprising a first unique genetic modification; (b-2) transfecting into the first plurality of gene edited cells a first plurality of recombinant polynucleotides, each recombinant polynucleotide in the first plurality comprising a first unique polynucleotide barcode sequence, thereby forming a first plurality of gene edited and barcoded cells, each gene edited and barcoded cell comprising the first unique polynucleotide barcode inserted into a barcode locus and the first unique genetic modification; (c) contacting said first plurality of gene edited and barcoded cells with a second gene editing agent thereby producing a second plurality of gene edited cells each comprising a second unique genetic modification; and (d) transfecting into the second plurality of gene edited cells a second plurality of recombinant polynucleotides, each recombinant polynucleotide in the second plurality comprising a second unique polynucleotide barcode sequence, thereby forming a second plurality of gene edited and barcoded cells, such that the second unique polynucleotide barcode is inserted into a barcode locus of each of said second plurality of gene edited and barcoded cells, each gene edited and barcoded cell comprising the second unique polynucleotide barcode inserted into a barcode locus and the second unique genetic modification, thereby producing a plurality of genetically engineered cells; wherein the method provides linking of unique barcodes sequentially integrated adjacent to one another at specific time points to enable lineage tracking and/or enables higher order combinatorial genetic modifications to be constructed and tracked with either a single barcode or short barcode array, and wherein multiple individual barcodes combine to generate a larger single barcode, where order of barcodes represents order of addition of genome edits, enabling sequencing-based approaches to identify and count barcode permutations or combinations.

2 . The method of claim 1 , further comprising repeating step (b-1) or steps (c) and (d) one or more times, wherein each repeat of step (b-1) or steps (c) and (d) employs a plurality of recombinant polynucleotides, each recombinant polynucleotide comprising a unique polynucleotide barcode sequence.

3 . The method of claim 1 , wherein
i) the first unique polynucleotide barcode of step (a-1) or step (b-2) is associated with the first unique genetic modification in each of said first plurality of gene edited and barcoded cells; ii) the second unique polynucleotide barcode of step (a-2) or step (d) is associated with the second unique genetic modification in each of said second plurality of gene edited and barcoded cells; iii) the barcode locus and at least a portion of the genome that includes a genomic sequence comprising the first unique genetic modification of each of said first plurality of gene edited and barcoded cells are sequenced, such that the first unique polynucleotide barcode is associated with the first unique genetic modification in a database; iv) the barcode locus and at least a portion of the genome that includes a genomic sequence comprising the second unique genetic modification of each of said second plurality of gene edited and barcoded cells are sequenced, such that the second unique polynucleotide barcode is associated with the second unique genetic modification in a database; v) each unique polynucleotide barcode is added adjacent to a most recently integrated unique polynucleotide barcode in the barcode locus; vi) each unique polynucleotide barcode is added upstream of a most recently integrated unique polynucleotide barcode in the barcode locus; vii) each unique polynucleotide barcode is added downstream of a most recently integrated unique polynucleotide barcode in the barcode locus; viii) each unique polynucleotide barcode is flanked on the recombinant polynucleotide by a right homology arm and/or a left homology arm; ix) each first unique polynucleotide barcode is flanked on each recombinant polynucleotide of the first plurality of polynucleotides by a first right homology arm, a second right homology arm, and a left homology arm, such that the first right homology arm and the left homology arm are homologous to a sequence at the barcode locus; and/or x) each unique polynucleotide is inserted in the barcode locus by homologous recombination or non-homologous end joining, or using an integrase.

4 . The method of claim 1 , wherein
i) each recombinant polynucleotide further comprises a marker polynucleotide and/or a donor polynucleotide; and/or ii) each recombinant polynucleotide is provided by a vector.

5 . The method of claim 1 , wherein
i) at least one of the first gene editing agent and the second gene editing agent is a meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector-based nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR) system, RNA-guided nuclease, RNA-guided nickase, a chemical agent, a recombinase, an integrase, or a transposase; ii) the first gene editing agent and the second gene editing agent are the same; iii) the first gene editing agent and the second gene editing agent are different; and/or iv) at least one of the first gene editing agent and the second gene editing agent is a meganuclease, an RNA-guided nuclease, or an RNA-guided nickase.

6 . The method of claim 1 , further comprising
i) sequencing from a sample of cells at least a portion of the chromosome that includes a genomic sequence comprising the second unique genetic modification from the second plurality of gene edited and barcoded cells; ii) inserting a new unique polynucleotide barcode in the barcode locus of each cell, wherein the barcode locus already comprises at least two unique polynucleotide barcodes prior to insertion of the new unique polynucleotide barcode, and sequencing the barcode locus such that the new unique polynucleotide barcode is associated with the at least two unique polynucleotide barcodes; iii) contacting said second plurality of gene edited and barcoded cells with a second nucleic acid sequence encoding a guide RNA capable of hybridizing with the recombinant polynucleotide, wherein the guide RNA forms a complex with a nuclease in each cell such that the guide RNA-nuclease complex cleaves the recombinant polynucleotide or the barcode locus.

7 . The method of claim 1 , wherein
i) the barcode locus is a chromosomal barcode locus; ii) the genetic modification is a designed genetic modification; and/or iii) the vector is removed f

Assignees

• Univ Leland Stanford Junior
• Government Of The Us Secretary Of Commerce

Inventors

• Salit Marc
• Steinmetz Lars M
• St Onge Robert
• Smith Justin D
• Roy Kevin R

Classifications

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2521/301

  Endonuclease · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/11 Primary

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title