Therapies that target the function of immune cells have signi cant clinical e cacy in diseases such as cancer and autoimmunity.Although functional genomics has accelerated therapeutic target discovery in cancer, its use in primary immune cells is limited because vector delivery is ine cient and can perturb cell states.Here we describe CHIME: CHimeric IMmune Editing, a CRISPR-Cas9 bone marrow delivery system to rapidly evaluate gene function in innate and adaptive immune cells in vivo without ex vivo manipulation of these mature lineages.This approach enables e cient deletion of genes of interest in major immune lineages without altering their development or function.We use this approach to perform an in vivo pooled genetic screen and identify Ptpn2 as a negative regulator of CD8+ T cell-mediated responses to LCMV Clone 13 viral infection.These ndings suggest that this genetic platform can enable rapid target discovery through pooled screening in immune cells in vivo.
Cre recombinase is widely used in a variety of organisms to manipulate DNA both in vitro and in vivo.Combined with its consensus DNA sequences, named loxP, it allows the DNA modi cation including deletions, insertions, translocations and inversions at speci c DNA locus of cells.To promote its use in plant cultured cells, we report here a detailed protocol for direct protein delivery of Cre recombinase into cultured Arabidopsis thaliana cells with an intact cell wall.Electroporation with the optimized buffer and program enables the e cient delivery of Cre protein and following recombination in cultured Arabidopsis thaliana cells.Speci cally, we describe the puri cation of Cre protein, the preparation of electroporation samples, and the assessment of electroporated cells.This simple, economical, and effective technology will contribute to the biological analysis of genes and cellular functions in cultured Arabidopsis thaliana cells.This protocol accompanies
