Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages

Abstract

Genome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral transduction. However, cell types derived from the differentiation of induced Pluripotent Stem Cells (iPSC), which often represent more relevant, patient-derived models for human pathology, are much more difficult to engineer as CRISPR/Cas9 delivery to these differentiated cells can be inefficient and toxic. Here, we present an efficient, lentiviral transduction protocol for delivery of CRISPR/Cas9 to macrophages derived from human iPSC with efficiencies close to 100%. We demonstrate CRISPR/Cas9 knockouts for three nonessential proof-of-concept genes—HPRT1, PPIB and CDK4. We then scale the protocol and validate for a genome-wide pooled CRISPR/Cas9 loss-of-function screen. This methodology enables, for the first time, systematic exploration of macrophage involvement in immune responses, chronic inflammation, neurodegenerative diseases and cancer progression, using efficient genome editing techniques.

Macrophages Derived From Human Induced Pluripotent Stem Cells: The Diversity of Protocols, Future Prospects, and Outstanding Questions

“…Because iMphs represent a homogeneous, standardizable, and genetically editable population, they provide a unique opportunity to study Mφ biology, including the role of specific individual genes in cell functionality, like it was recently done by several groups ( Zhang et al, 2017 ; Hall-Roberts et al, 2020 ; Navarro-Guerrero et al, 2021 ).…”

Optimizing the Method for Differentiation of Macrophages from Human Induced Pluripotent Stem Cells

“…Macrophages derived from human embryonic stem cells have the same functions as macrophages from peripheral blood and bone marrow monocytes, but there are ethical issues involved [5]. hiPSCs become a favorable choice, because they have no ethical limitations and can be easily manipulated by genome editing technology [6][7][8][9].…”

iPSC-Derived Natural Killer Cell Therapies - Expansion and Targeting

“…Genetic engineering approaches such as transposons and lentiviral delivery ensure efficient transgene insertion and stable expression in iPSCs (58,59). TALENS and CRISPR/Cas9 can also be used for more precision in knocking in or deleting specific genes (60)(61)(62)(63)(64). Once engineered, the engineered and undifferentiated iPSCs can be frozen and stored to allow for consistent production of NK cells with an identical phenotype.…”