Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for modeling competition of multiple cancer mutations in vivo
The invention involves inducing 3-50 or more mutations (e.g., any whole number between 3 and 50 of mutations, with it noted that in some embodiments there can be up to 16 different RNA(s), e.g., sgRNAs each having its own a promoter, in a vector, such as AAV, and that when each sgRNA does not have its own promoter, there can be twice to thrice that amount of different RNA(s), e.g., sgRNAs, e.g., 32 or even 48 different guides delivered by one vector) in transgenic Cas9 eukaryotes to model genetic disease, e.g. cancer. The invention comprehends testing putative treatments with such models, e.g., testing putative chemical compounds that may be pharmaceutically relevant for treatment or gene therapy that may be relevant for treatment, or combinations thereof. The invention allows for the study of genetic diseases and putative treatments to better understand and alleviate a genetic disease or a condition, e.g., cancer.
1. A method comprising:
(a) delivering a vector expressing a plurality of RNAs to guide Cas9 to a plurality of target loci into a non-human mammal having cells that express or that are able to be induced to express or that conditionally express Cas9,
thereby introducing 3 or more mutations in the plurality of target loci in the non-human mammal and causing a genetic disorder in the non-human mammal,
wherein the vector comprises (i) a donor polynucleotide, (ii) coding sequences for the plurality of RNAs, (iii) a coding sequence for a Cre recombinase, and (iv) a reporter gene; and
(b) identifying, among the introduced 3 or more mutations, a combination of mutations that results in the genetic disorder.
2. The method of claim 1 , wherein the method comprises delivering to cells of the non-human mammal the vector, and the non-human mammal is a mouse having cells that express or that are able to be induced to express or that conditionally express Cas9.
3. The method of claim 2 , wherein the mouse having cells that express Cas9 comprises a mouse that has had a Cas9 transgene knocked into the Rosa26 locus, wherein the Cas9 transgene further comprises a Lox-Stop-polyA-Lox (LSL) cassette thereby rendering Cas9 expression inducible by Cre recombinase.
4. The method of claim 1 , wherein one or more of the plurality of mutations are or have been correlated to a genetic disease or cancer.
5. The method of claim 4 , further comprising modeling the genetic disease or cancer.
6. The method of claim 4 , further comprising testing treatment(s) for the genetic disease on the cells to which the vector has been delivered in a subject.
7. The method of claim 6 , further comprising treating the subject based on results from the testing of treatment(s).
8. The method of claim 4 , wherein the genetic disease or cancer is lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, acute myeloid leukemia, skin basal cell carcinoma, bladder cancer, breast cancer, carcinoid cancer, chronic lymphocytic leukemia, colorectal cancer, lymphoma, diffuse large B-cell lymphoma, endometrial cancer, esophageal cancer, esophageal adenocarcinoma, glioblastoma multiforme, glioma, head and neck cancer, kidney cell cancer, medulloblastoma, melanoma, multiple myeloma, nasopharyngeal cancer, neuroblastoma, ovarian cancer; prostate cancer, rhadbdoid tumor, thyroid cancer, or urinary bladder cancer.
9. The method of claim 1 , wherein the plurality of mutations comprise specific mutations or precise sequence substitutions in the plurality of targeted loci.
10. The method of claim 1 , wherein the 3 or more mutations comprise at least one loss-of-function mutation and at least one gain-of-function mutation.
11. The method of claim 1 , wherein the vector is an adeno-associated virus vector.