Dataset Information

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.

SUBMITTER: Navarro-Guerrero E

PROVIDER: S-EPMC7895961 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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

Navarro-Guerrero Elena E Tay Chwen C Whalley Justin P JP Cowley Sally A SA Davies Ben B Knight Julian C JC Ebner Daniel D

Scientific reports 20210219 1

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 dif ...[more]

PMID: 33608581

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Project description:OBJECTIVE:To gain mechanistic insights into the role of LIPA (lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages. APPROACH AND RESULTS:We used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technology to knock out LIPA in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage LIPA loss-of-function phenotypes. LIPA was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of LIPA (LIPA-/-) had barely detectable LAL enzymatic activity. Control and LIPA-/- IPSDM were loaded with [3H]-cholesteryl oleate-labeled AcLDL (acetylated low-density lipoprotein) followed by efflux to apolipoprotein A-I. Efflux of liberated [3H]-cholesterol to apolipoprotein A-I was abolished in LIPA-/- IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [3H]-cholesterol-labeled AcLDL, [3H]-cholesterol efflux was, however, not different between control and LIPA-/- IPSDM. ABCA1 (ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and LIPA-/- IPSDM. In nonlipid loaded state, LIPA-/- IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and LIPA-/- IPSDM. LIPA-/- did not impact lysosomal apolipoprotein-B degradation or expression of IL1B, IL6, and CCL5. CONCLUSIONS: LIPA-/- IPSDM reveals macrophage-specific hallmarks of LIPA deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease-associated LIPA genetic variation in human macrophages.

Dataset Information

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

Publications

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

Similar Datasets

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