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Guide Swap enables genome-scale pooled CRISPR–Cas9 screening in human primary cells

Ting, Pamela and Lee, Scott and Parker, Albert and Trussell, Christopher and Sharif, Orzala and Luna, Fabio and Federe, Glenn and Barnes, Whitney and Walker, John and Vance, Julie and Gao, Mu-Yun and Klock, Heath and Clarkson, Scott and Russ, Carsten and Miraglia, Loren and Cooke, Michael and Boitano, Anthony and Mcnamara, Peter and Lamb, John and Schmedt, Christian and Snead, Jennifer (2018) Guide Swap enables genome-scale pooled CRISPR–Cas9 screening in human primary cells. Nature Methods, 15 (11). pp. 941-946. ISSN 15487105

Abstract

CRISPR–Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells—an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR–Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.

Item Type: Article
Date Deposited: 17 Jan 2019 00:45
Last Modified: 17 Jan 2019 00:45
URI: https://oak.novartis.com/id/eprint/36667

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