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. 2021 Feb 19;11:4245. doi: 10.1038/s41598-021-82137-z

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Lentiviral transduction for the delivery of CRISPR/CAS9 to macrophages derived from human iPSC. Embryoid bodies were generated from human iPSC in the presence of specific growth factors. The embryoid bodies were then differentiated in the presence of M-CSF and IL-3 until the emergence of precursor macrophages. Precursor macrophages were transduced with either lentivirus containing individual sgRNAs or the TKOv3 CRISPR lentiviral library in the presence of VPX-VLPs. For precursor macrophages transduced with individual guides, puromycin selection was started 3 days post-transduction and cells were allowed to differentiate to macrophages in M-CSF for another 11 days. For the precursor macrophages transduced with the TKOv3 library, cells were differentiated to macrophages in the presence of M-CSF for 14 days, after which the cells were collected for the Reference sample, genomic DNA extracted and analysed by next-generation sequencing. This protocol enables future application for genome-wide screens, for example based on phenotypic selection in the presence of the TKOv3 CRISPR lentiviral library (indicated by dashed lines).