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
This protocol describes a reproducible methodology to sample vegetation canopy height and aboveground biomass in low stature ecosystems, using observations collected with a lightweight Unmanned Aerial Vehicle \(UAV or 'drone'), RTK-GNSS and destructive harvesting of vegetation.
Ubiquitination is an essential protein modi cation that in uences multiple eukaryotic processes such as substrate degradation, pathway alterations.However, because of the interaction between the substrate and other proteins, it is di cult to detect the exact ubiquitination level of a speci c protein using immunoprecipitation and western blot method in a substrate-overexpression cell line.In our nature paper, we used a denaturing conditions to eliminate proteins conjugated with haemagglutinin-tagged PD-1.By using protocol, we can detect the exact ubiquitination level of PD-1 with western blot analysis.After cells have been collected, the described protocol can be completed in 2-3 d.
This protocol describes a method to site-speci cally cleave proteins using chemicals upon insertion of the appropriate tag into the cleaving proteins.It can be applied to water soluble as well as membrane proteins with e ciency comparable to enzymes.The fact that chemicals can be used to replace enzymes in this method highlights the unique aspects of this technology.The cleavage can be achieved within a day upon obtaining target proteins.Detailed technical aspects of this method will be discussed in this protocol.
microRNAs \(miRNAs) are small noncoding RNAs that play important regulatory roles in plants, animals and viruses.However, measuring miRNA activity in vivo remains a big challenge.In this protocol, using a miRNA-mediated sgRNA releasing strategy and dCas9-VPR to drive a transgene RFP expression, we create a miRNA sensor that can faithfully measure miRNA activity at cellular levels.When sgRNAs are designed to target endogenous locus, we show this system can be adapted to achieve cell type speci c activation of endogenous genes.Furthermore, when dCas9 is fused with a transcriptional repressor or a base editor, we show this system can be used to repress the expression of endogenous genes or mutate speci c DNA bases of chromosome upon induction by cell type-speci c miRNAs.This step-by-step protocol is related to the publication "A microRNA-inducible CRISPR-Cas9 platform serves as microRNA sensors and cell type speci c genome regulation tools" in Nature Cell Biology.
TCR ligand discovery is essential for elucidating targets of anti-tumor immunity and designing targeted immunotherapies.Here, we describe a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis.
Our method for analyzing histone modifications, scChIC-seq (single-cell chromatin immunocleavage sequencing), involves targeting of the micrococcal nuclease (MNase) to a histone mark of choice by tethering to a specific antibody. Cleaved target sites are then selectively PCR amplified. We show that scChIC-seq reliably detects H3K4me3 and H3K27me3 target sites in single human white blood cells. The resulting data are used for clustering of blood cell types.
This Standard Operating Procedure \(SOP) describes the methodology for performing immunohistochemistry staining using para n-embedded or frozen tissue, or cytology samples
Here, we describe Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing \(ECCITEseq) for the high-throughput characterization of at least ve modalities of information from each single cell: transcriptome, immune receptor clonotypes, surface markers, sample identity and single guide RNA \(sgRNAs).ECCITE-seq adapts CITEseq and Cell Hashing to a 5' tag-based scRNA-seq assay, integrating clonotype and cell surface marker information to RNAbased cellular phenotypes in immune cells.Additionally, ECCITE-seq allows the direct detection of sgRNAs, through a minor modi cation to the scRNA-seq work ow, facilitating high throughput and sensitive single cell perturbation screens compatible with existing guide libraries and commonly used vectors.
