CRISPR mediated in vivo modeling and genetic screening of tumor growth and metastasis
The present invention relates to in vivo methods for modeling tumor formation and/or tumor evolution comprising the use of eukaryotic cells in which one or more genetic target locus has been altered by the CRISPR/Cas system, and which cells are transplanted in non-human eukaryote as a model system for tumor formation and tumor evolution. In particular in vivo genetic screening methods for identifying genes involved in tumorigenesis and metastasis are disclosed. The invention further relates to kits and components for practicing the methods, as well as materials obtainable by the methods, in particular tumor and metastasis samples and cells or cell lines derived therefrom. The invention also relates to diagnostic and therapeutic methods derived from the information obtained in the modeling methods.
1. A method for identifying genes implicated in the metastasis of a primary tumor to the lung comprising the steps of:
a) transducing a population of Cas9 expressing immortalized eukaryote cells with a genome-wide library of guide RNAs to inactivate one or more genetic target loci in each cell by CRISPR-cas9 gene editing;
b) transplanting the modified, gene-edited cell population into a non-human animal host
c) isolating guide RNAs enriched in tumors in the lung that have metastasized from primary tumors obtained from the transplanted cell population; and
d) identifying candidate genes implicated in the metastasis of primary tumor cells to the lung,
wherein said candidate genes comprise at least one of Nf2, Pten, Cdkn2a, Trim72, Fga, miR-345, and miR-152,
wherein a loss of function mutation in any one of the candidate genes alone is sufficient to accelerate the rate of metastasis formation to the lung relative to untransduced and nontargeting sgRNA controls, and
wherein the modified, gene-edited cell population implanted into the non-human animal host does not metastasize to the liver, kidney, or spleen.
2. The method of claim 1 , further comprising
a) transducing a second population of Cas9 expressing immortalized eukaryote cells with guide RNAs targeting the candidate genes identified in step d) using CRISPR-Cas9 gene editing;
b) implanting the gene-edited cell population into the non-human animal host, and
c) isolating guide RNAs enriched in tumors in the lung that have metastasized from primary tumors obtained from the transplanted cell population, thereby validating the involvement of the candidate genes in the metastasis to the lung, wherein the modified, gene-edited cell population implanted into the non-human animal host does not metastasize to the liver, kidney, or spleen.
3. The method of claim 1 , wherein the gene-edited cell population is implanted subcutaneously.
4. The method of claim 1 , wherein the non-human animal host is immunocompromised.
5. The method of claim 1 , wherein said candidate genes comprise Nf1 or Pten.
6. The method of claim 1 , wherein said candidate genes comprise at least three genes chosen from Nf2, Pten, and any one of Cdkna2a, Trim72, Fga, miR-345, and miR-152.
7. The method of claim 1 , wherein said candidate genes comprise at least three genes chosen from Nf2, Pten, any two of Cdkna2a, Trim72, Fga, miR-345, and miR-152.
8. The method of claim 1 , wherein said candidate genes comprise Nf2, Pten, Cdkna2a, Trim72, Fga, miR-345, and miR-152.
9. The method of claim 1 , wherein said candidate genes further comprise Ube2g2, Ptges2 or ATP-dependent DNA Ligase IV (Lig4).
10. The method of claim 1 , wherein, prior to modification by gene editing, the immortalized eukaryote cells comprise a loss-of-function mutation in a tumor suppressor gene and a gain-of-function mutation in a proto-oncogene.
11. The method of claim 10 , wherein the tumor suppressor gene comprises a p53−/− and/or Lkb1−/−.
12. The method of claim 10 , wherein the proto-oncogene comprises Kras.
13. The method of claim 12 , wherein the Kras gain-of-function mutation comprises a KrasG12D/+ oncogenic mutation.
14. The method of claim 1 , wherein the unmodified population of immortalized eukaryote cells does not metastasize to the lung.
15. The method of claim 1 , wherein the population of immortalized eukaryote cells is obtained from a lung cancer.
16. The method of claim 1 , wherein the population of immortalized eukaryote cells is a non-small-cell lung cancer (NSCLC) cell line.
17. The method of claim 1 , wherein the immortalized eukaryote cell is a cell from an eukaryote selected from the group consisting of a mammal, a primate, a rodent, a mouse, a rat, a rabbit, a canine, a dog, a cow, a bovine, a sheep, an ovine, a goat and a pig.
18. The method of claim 1 , wherein said Cas9 is a Cas9 originating from Streptococcus pyogenes, Streptococcus thermophiles, or Staphylococcus aureus ; a mutated Cas9 having an altered catalytic activity; a catalytically inactive Cas9; or wherein the Cas9 is fused to an enzyme which modifies genomic DNA or genomic DNA architecture or a polypeptide which alters gene transcription.