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

Ting PY, Parker AE, Lee JS, Trussell C, Sharif O, Luna F, Federe G, Barnes SW, Walker JR, Vance J, Gao MY, Klock HE, Clarkson S, Russ C, Miraglia LJ, Cooke MP, Boitano AE, McNamara P, Lamb J, Schmedt C, Snead JL.
Source: Nat Methods
Publication Date: (2018)
Issue: 15(11):
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
Species: mouse
Tissue Origin: colon
4D-Nucleofector® X-Unit

CT26, human T cells and human CD34 have been virally transduced and 2-3 days later electroporated with the 4D Nucleofector, gRNA and RNPs. For transfection of all primary cells, CM-137 and P3 were applied. The CT26 wells with SE kit and DS-120.


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.