Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells.

Optimizing CRISPR/Cas9 genome editing in primary human hematopoietic cells to advance studies into HIV biology

Co-delivery of lentiviral vectors and Cas9-containing virus-like particles enables rapid, scalable manufacture of gene-edited CAR T cells

Chimeric antigen receptor T cell immunotherapies are transforming therapies for hematological malignancies and solid tumors and can be enhanced by targeted gene knockout. Here, we report lentiviral-based virus-like particles that package and deliver Cas9 ribonucleoproteins to primary human T cells. Using distinct pseud...

Production of virus-like particles with AsCas12a nuclease and CMV-driven crRNA for mammalian genome editing

A detailed protocol is described for a small-scale production of AsCas12a-VLPs using three distinct transfection methods and a large-scale production of VLPs using calcium-phosphate transfection, showing that both production scales yield comparable nuclease loading into VLPs and similar editing efficiencies.

A lipid nanoparticle platform for high yield CRISPR-targeted homology directed repair enables fully non-viral CAR T cell generation

Genome-wide CRISPR screening identifies cellular factors controlling nonviral genome editing efficiency

To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion incre...

Genome-Wide CRISPR Screening Identifies Cellular Factors Controlling Nonviral Genome Editing Efficiency

A novel genome-wide CRISPR screening strategy that will facilitate the systematic engineering of novel nonviral genome editing delivery methods, where the identified novel gene hits can be further used to increase editing efficiency for other therapeutically relevant cell types.