BARCODED CLONAL TRACKING OF GENE TARGETING IN CELLS
Methods and compositions for monitoring a plurality of independent genomic modifications in cell lineages are provided.
1 . A method of tracking cell populations comprising an introduced DNA molecule, the method comprising
introducing a plurality of homology recombination donor template polynucleotide sequences into a plurality of cells under conditions such that at least part of the homology recombination donor template polynucleotide sequences are introduced into a target genomic sequence of a cell from the cell population, wherein the homology recombination donor template polynucleotide sequences comprise in the following order: a left homology arm, a coding sequence, and a right homology arm, wherein
(1) the coding sequence comprises a silent mutation compared to a wildtype coding sequence of the cell, wherein the plurality of homology recombination donor template polynucleotide sequences comprises different silent mutations and wherein at least two cells receive recombined polynucleotides, each having a different silent mutation; or
(2) between the left and right homology arms and outside the coding sequence a barcode sequence is present, wherein the plurality comprises different barcodes and wherein at least two cells receive recombined polynucleotides, each having a different barcode sequence.
2 . The method of claim 1 , wherein the plurality of homology recombination donor template polynucleotide sequences comprises at least 10 different silent mutations and wherein at least 10 cells receive recombined polynucleotides, each having a different silent mutation
3 . The method of claim 1 , wherein the plurality comprises at least 10 different barcodes and wherein at least 10 cells receive recombined polynucleotides, each having a different barcode sequence.
4 . The method of claim 1 , wherein between the left and right homology arms and outside the coding sequence the barcode sequence is present and wherein following the coding sequences there is a polyA sequence and the barcode is present between the polyA sequence and the right homology arm.
5 . The method of claim 1 , wherein the cells are primary cells.
6 . The method of claim 5 , wherein the cells are primary hematopoietic cells.
7 . The method of claim 6 , wherein the cells are primary hematopoietic stem cells.
8 . The method of claim 6 , wherein the cells are primary T-cells.
9 . (canceled)
10 . The method of claim 1 , wherein the introducing comprises providing a targeted nuclease into the cell wherein the targeted nuclease introduces a double-stranded break in the genomic DNA of the cell at a sequence in the genome to which the right and left homology arm sequences have homology.
11 . The method of claim 10 , wherein the targeted nuclease is targeted by a single guide RNA (sgRNA).
12 . The method of claim 11 , wherein the sgRNA comprises one or more modified nucleotides.
13 . The method of claim 11 , wherein the targeted nuclease comprises CRISPR-associated protein (Cas) polypeptide.
14 . The method of claim 10 , wherein the targeted nuclease comprises a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) or a meganuclease.
15 . The method of claim 1 , wherein the introducing comprises introducing adeno-associated viral (AAV) vectors comprising the homology recombination donor template polynucleotide sequences.
16 . The method of claim 15 , wherein the introducing further comprises introducing into the cells a ribonucleoprotein (RNP) comprising a single guide RNA (sgRNA) and a CRISPR-associated protein (Cas) polypeptide.
17 . The method of claim 1 , further comprising allowing the cell population to divide thereby forming an expanded cell population; and
sequencing recombined polynucleotides from the expanded cell population, thereby allowing for tracking of different cells based on the different silent mutations or different barcodes.
18 . The method of claim 17 , wherein the cells are primary hematopoietic cells and the allowing comprises introducing the cells into an animal and the cells divide and optionally differentiate in the animal.
19 - 21 . (canceled)
22 . The method of claim 1 , wherein the coding sequence encodes hemoglobin (HBB), Wiskott-Aldrich Syndrome Protein (WAS), Iduronidase (IDUA), Interleukin-7 receptor alpha (Il7RA), Interleukin-2 receptor gamma chain (Il2RG), gp91phox (CYBB), V(D)J recombination-activating protein 1(RAG), V(D)J recombination-activating protein 2 (RAG2), Galactosylceramidase (GALC), Tripeptidyl-peptidase 1(TPP), Glucosylcermidase beta (GBA), Cystic Fibrosis Transmembrane Receptor (CFTR), Forxhead box protein P3 (FOXP3), CD40 Ligand (CD40L), Perforin 1 (PRF1), T-cell Receptor (TCR), Beta-2-microglobulin (B2M), ATP-binding cassette sub-family D member1 (ABCD-1), Brain-derived neurotrophic factor (BDNF), or phenylalanine hydroxylase (PAH).
23 . (canceled)
24 . A plurality of homology recombination donor template polynucleotide sequences comprising in the following order: a left homology arm, a coding sequence, and a right homology arm, wherein
(1) the coding sequence comprises a silent mutation compared to a wildtype coding sequence, wherein the plurality comprises at least two different silent mutations; or
(2) between the left and right homology arms and outside the coding sequence a barcode sequence is present, wherein the plurality comprises at least two different barcodes.
25 - 46 . (canceled)