CD300ld regulates tumor immunity - In vivo Crispr-Cas9 screening

To identify surface proteins on myeloid cells involved in the establishment of the tumor immune microenvironment, we selected transmembrane protein encoding genes with a high myeloid/lymphoid expression ratio to construct a sgRNA library. The sgRNA pool was transduced into hematopoietic stem/progenitor cells of Cas9 mice to transplant the lethal irradiated mice for immune system reconstitution, then followed by B16-F10 tumor inoculation. Once the tumor was established, the tumor and bone marrow (BM) were collected for sgRNA deep sequencing and comparison. Lentiviral particles encoding sgRNA pool were transduced into freshly lineage negative HSC/progenitors isolated from fetal liver of Cas9-tdTomatao mice at a multiplicity of infection of 0.25. At 60 h post spin-infection, the transduced cells were retro-orbitally transplanted in lethally irradiated (X-ray, 9.5 gray) recipient mice (1 million cells per mouse). B16-F10 cells were injected s.c to recipient mice 4 weeks after transplantation. Mice were sacrificed at day-15 post tumor cell inoculation, and CD45+ cells were collected from bone marrow and tumor by flow cytometry. genomic DNA was isolated from all samples using Universal Genomic DNA Purification Mini Spin Kit (Qiagen). sgRNA encoding fragments were amplified for deep sequencing .