Methods and compositions for studying cell evolution
AssigneesThe Broad Institute, Inc., Dana-Farber Cancer Institute, Inc., Massachusetts Institute of Technology
InventorsPratiti Bandopadhayay, Rameen Beroukhim, Paul Blainey, David Feldman, Cory Johannessen, Funien Tsai
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Abstract
The subject matter disclosed herein is generally directed to methods and compositions for tagging cells of interest, tracking evolution of the tagged cells, and recovering the original tagged cells for further study. Specifically, cells are tagged with a DNA construct encoding a barcode sequence comprising a guide sequence. Barcoded cells can then be recovered using a reporter construct having CRISPR target sequences specific for the cell having a barcode of interest.
Claims20
An isolated subpopulation of originating cells of an originating cell population, wherein:
- (A) each originating cell of the originating cell population comprises DNA construct comprising a unique barcode sequence integrated into the genome of each originating cell, wherein:
- (i) progeny cells of each originating cell of the originating cell population comprise the same barcode;
- and (ii) each barcode sequence comprises a guide sequence configured to guide a CRISPR-Cas effector protein to one or more target loci of a barcode sequence-matched reporter construct, wherein the barcode sequence is configured to introduce an insertion or deletion or a functional domain to the barcode sequence-matched reporter construct, thereby activating at least one of the one or more selection markers or reporters of the barcode sequence-matched reporter construct;
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- (B) the isolated subpopulation of originating cells comprises the barcode sequence-matched reporter construct for one or more selected unique barcode sequences of the originating cell population and expresses the activated at least one of the one or more selection markers or reporters of the barcode sequence-matched reporter construct.
An isolated subpopulation of originating HeLa cells of an originating HeLa cell population, wherein:
- (A) each originating HeLa cell of the originating HeLa cell population comprises a DNA construct comprising a unique barcode sequence integrated into the genome of each originating HeLa cell, wherein:
- (i) progeny cells of each originating HeLa cell of the originating HeLa cell population comprise the same barcode;
- and (ii) each barcode sequence comprises a guide sequence configured to guide a CRISPR-Cas effector protein to one or more target loci of a barcode sequence-matched reporter construct, wherein the barcode sequence is configured to introduce an insertion or deletion or a functional domain to the barcode sequence-matched reporter construct, thereby activating at least one of the one or more selection markers or reporters of the barcode sequence-matched reporter construct;
- and
- (B) the isolated subpopulation of originating HeLa cells comprises the barcode sequence-matched reporter construct for one or more selected unique barcode sequences of the originating HeLa cell population and expresses the activated at least one of the one or more selection markers or reporters of the barcode sequence-matched reporter construct.
An isolated subpopulation of originating cells of an originating cell population, wherein:
- (A) each originating cell of the originating cell population comprises a DNA construct comprising a unique barcode sequence integrated into the genome of each originating cell, wherein:
- (i) progeny cells of each originating cell of the originating cell population comprise the same barcode;
- and (ii) each barcode sequence comprises a guide sequence configured to guide a CRISPR-Cas effector protein to a target locus of a barcode sequence-matched reporter construct, wherein the sequence of the target locus is configured such that introducing an insertion or deletion in said target sequence creates a frameshift in said open reading frame, whereby one or more markers are activated or inactivated, and wherein the one or more markers comprise a first fluorescently detectable marker detectable at a first wavelength, and a second fluorescently detectable marker detectable at a second wavelength;
- and
- (B) the isolated subpopulation of originating cells comprises the barcode sequence-matched reporter construct for one or more selected unique barcode sequences of the originating cell population and expresses the activated one or more markers of the barcode sequence-matched reporter construct, wherein the first fluorescently detectable marker or the second fluorescently detectable marker identifies the isolated subpopulation of originating cells for purification.
Dependencies are inferred from the claim text.