CRISPR-based functional genomics in human dendritic cells

• Dendritic cells regulate processes from antitumour and antiviral immunity to host-micrbobe communication on mucous surface → hard to manipulate DCs, limits ability to probe specific immune responses
• develop CRISPR-Cas9 genome editing method for human monocyte-derived DCs → median efficiency of knockouts at 94% across 300 genes
  • genetic screens performed on moDCs, identify mechanisms by which the DCs tune respones to lipopolysaccharide from human microbiome
  • lipopolysaccharides: also known as endotoxins: consist of lipid and polysaccharide and present in bacterial cell wal and extremeley very toxic →
    • endotoxin kept within bacterial cell and released after destruction of bacterial cell wall (does not necessarily require destruction of bacterial cell wall but excreted through bacterial membrane vessicles)
    • LPS makes up majority of outer membrane and contributes to structural integrity of bacteria
    • protects bacterial membrane from certain attacks, increases negative charge of membrane and helps stabilize structure → look into O-antigen, these attract DCs but more importantly, LPS in gram-negative bacteria make it a great target for antimicrobial therapy
  • THIS ALSO CREATES AUTOIMMUNE RESPONSE → we have LPS in human microbiome to give bacteria structure and protect it from chemical attacks
• moncyte-derived DCs → white blood cells
• donor-specific responses to lipopolysaccharaides also worked, allows for identifying candidate genes that control specificity per patient which can identify determinants in variation in immunity

Intro:

• DCs: detect invaders and initiate innate immune response to clear them, they also initiate adaptive immune responses that are antigen-speicfic and tailored to context in which antigen was detected

• mediate pathogen clearent, tumour cell killing, and tolernace to microbiome bacteria or dietary antigens → shape host-pathogen and host-microbiome interactions and in etiology of autoimmune disorders and can play a critical role in develpoing next generation of immunotherapies

• dissecting pathways for immune stimuli → adaptive immune response is blocked by difficulties in genetically manipulating human DCs

  • gene expression in human DCs by RNAi hve helped, suffers from limited efficacy and specificity

• one way to address this is to knock out genes in DC precurosr populations → hard to screen and susceptible to batch effects

• do not permit probing the functions of genes required for DC differentiation and culture → fail to capture variation in immune phenotypes

• NEW METHOD: Crispr-Cas9 strategy used to construct targeted knock-outs direcrly in human moncyte-dervied DCs which are readily derived from donor blood and used for research and clinical applications

  1. conduct genetic screen for factors that recognize innate immune ligand from human microbiome, LPS from gut B.theta, reacpitulating known LPS signaling pathways, and revealing candidate genes that mediate specific-specific LPS recog and give rise to inter-individual variaion in response to LPS
  2. makes it easier for identifying receptors for innate immune ligans and pinpointing genetic bases of inter-individual variation in human immunity

• To enable introduction of knockouts, non-viral genome editing strategy developed based on electroporation of in-vitro assembled Cas9-sgRNA complexes