Delivery, use and therapeutic applications of the CRISPR-cas systems and compositions for modeling mutations in leukocytes
The invention involves a method for modulating leukocyte activity, comprising delivering to a leukocyte a vector containing nucleic acid molecule(s), whereby the leukocyte contains Cas9 and the vector expresses one or more RNAs to guide the Cas9 to introduce mutations in one or more target genetic loci in the leukocyte, thereby modulating expression of one or more genes expressed in the leukocyte. The invention also involves identifying genes associated with leukocyte responses and experimental modeling of aberrant leukocyte activation and diseases associated with leukocytes by introducing mutations into leukocytes. 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 leukocyte associated diseases.
1. A method of identifying a gene(s) or genetic element(s) capable of modulating a leukocyte immune response, comprising:
(a) obtaining a plurality of leukocytes from a mouse genetically modified to contain a Cas9 transgene expression cassette integrated into the Rosa26 locus of each cell, wherein said transgene is able to express Cas9 protein or is able to be induced to express or conditionally express Cas9 protein in said leukocytes;
(b) delivering in vitro to the plurality of leukocytes a plurality of different vectors containing nucleic acid molecule(s) encoding a plurality of Cas9 RNA guide sequences (i) to guide the Cas9 to introduce mutations in different target genetic loci or (ii) to guide the Cas9 to activate or inhibit expression of target gene(s) in the leukocytes, thereby modulating expression of different genes or genetic elements expressed in the leukocytes;
(c) stimulating in vitro the plurality of leukocytes under conditions to induce an immune response;
(d) detecting one or more markers associated with the induced immune response in the leukocytes;
(e) isolating or sorting leukocyte(s) according to expression levels of the one or more markers; and
(f) identifying the RNA guide sequences from the plurality of Cas9 guides sequences delivered in (b) that are encoded for by the nucleic acid molecule(s) in the isolated or sorted leukocytes, thereby identifying gene(s) or genetic elements associated with the one or more markers.
2. The method of claim 1 , wherein the genes or genetic elements are identified by sequencing one or more of the nucleic acid molecules present in the isolated or sorted leukocytes.
3. The method of claim 1 , wherein the leukocyte immune response is associated with activation, inhibition, exhaustion, differentiation, migration, adhesion, death, or proliferation of the stimulated plurality of leukocytes.
4. The method of claim 1 , wherein the one or more markers are selected from the group consisting of tumor necrosis factor alpha (TNF-α), CD86, interferon-γ (IFN γ), IL-4, TGFβ and IL-17.
5. The method of claim 1 , wherein the one or more markers are detected by Fluorescence Activated Cell Sorting (FACS).
6. The method of claim 1 , wherein the genetically modified mouse is a model as to a disease associated with leukocytes.