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.
Claims21
A polynucleotide reporter construct comprising one or more CRISPR-Cas guide molecule target sequences, 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.
A reporter system comprising:
- a) a polynucleotide reporter construct comprising one or more guide molecule 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;
- b) a CRISPR-Cas effector protein, or a nucleotide sequence encoding the CRISPR-Cas effector protein;
- c) a library comprising a set of guide molecule constructs each construct encoding a different guide sequence, the guide sequence comprising a barcode sequence and each guide sequence configured to guide the CRISPR-Cas effector protein to one of the one or more target loci of the polynucleotide reporter construct.
A method of 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 one or more 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;
- b) exposing the mixed population of cells to one or more perturbations;
- c) determining cells of interest by sequencing a portion of the mixed population of cells and assessing a ratio of the different barcode sequence counts;
- d) selecting the cells of interest by introducing polynucleotide reporter constructs comprising target loci for the guide sequences comprising the one or more 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 will guide the CRISPR-Cas effector protein to the target loci of the polynucleotide reporter construct, and wherein the CRISPR-Cas effector protein will make a frame shift edit at the target loci that shifts the first type of markers in frame such that the first type of one or more markers are expressed, and such that the second type of one or more markers are shifted out-of-frame such that second type of markers are no longer expressed;
- e) retrieving the cells of interest based on expression of the first type of one or more markers.
The construct, system, or method of any of the proceeding claims, wherein the first type and second type of markers are selectable markers, such as antibiotic resistance markers, affinity tags, optically-detectable markers, chemiluminescent detectable markers, fluorescently detectable markers, surface markers or a combination thereof.
The construct, system, or method of any of the proceeding claims, wherein the polynucleotide construct comprises an out-of-frame stop codon between the first type of marker and the second type of marker.
The construct, system, or method of any one of the proceeding claims wherein the polynucleotide reporter construct, the guide molecule construct, and/or the polynucleotide encoding the CRISPR-Cas protein are operably linked to a regulatory element.
The construct, system, or method of any of the proceeding claims, wherein the construct further encodes a stop codon upstream of the target loci.
A population of cells comprising a plurality of cells, each of the plurality of cells comprising a guide molecule construct from a set of guide molecule constructs, each construct encoding a different guide sequence, the guide sequence comprising a barcode sequence and each guide sequence configured to guide a CRISPR-Cas effector protein to one or more target loci of a reporter construct.
Dependencies are inferred from the claim text.