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.
Claims9
A method of recovering selecting one or more cells from mixed populations of cells comprising:
- a) tagging individual cells in a mixed population of cells with a guide molecule construct encoding a guide sequence from a library of constructs encoding different guide sequences, each guide sequence encoding a unique barcode sequence, and each guide sequence configured to guide a CRISPR-Cas effector protein to a target loci of a polynucleotide reporter construct, the polynucleotide reporter construct comprising the target loci, a first type of one or more markers that are out-of-frame, and a second type of one or more markers that are in frame,
- wherein one guide molecule construct is integrated into the genome of each cell, whereby the guide molecule construct is passed to its progeny upon propagation of the mixed population of cells;
- b) expanding the mixed population of cells for one or more generations, wherein each cell and the progeny of each cell in the mixed population of cells have the same guide molecule construct;
- c) splitting the mixed population of cells into one or more test populations and recovery populations;
- d) exposing the test population of cells to one or more perturbations;
- e) determining cells of interest comprising barcodes of interest by sequencing the test population of cells and assessing a ratio of the different barcode sequence counts;
- f) recovering the cells of interest by introducing to the recovery population polynucleotide reporter constructs comprising target loci for the guide sequences comprising the barcodes of interest and a CRISPR-Cas effector protein, or inducing expression within the cells of a CRISPR-Cas effector protein, wherein the guide sequence expressed in cells having the barcodes of interest guides the CRISPR-Cas effector protein to the target loci of the polynucleotide reporter construct, and wherein the CRISPR-Cas effector protein makes a frame shift edit at the target loci that shifts the first type of markers in frame wherein the first type of one or more markers are expressed, and wherein the second type of one or more markers are shifted out-of-frame wherein the second type of markers are no longer expressed;
- g) retrieving the cells of interest that express the first type of one or more markers.
Dependencies are inferred from the claim text.