A transient reporter for editing enrichment (TREE) in human cells.

David A Brafman,Grace Schwarz,Kylie Standage-Beier,Nicholas Brookhouser,Stefan J Tekel,Toan Nguyen,Xiao Wang

doi:10.1093/nar/gkz713

David A Brafman, Grace Schwarz + Show 5 more

Open Access

https://doi.org/10.1093/nar/gkz713

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Abstract

Current approaches to identify cell populations that have been modified with deaminase base editing technologies are inefficient and rely on downstream sequencing techniques. In this study, we utilized a blue fluorescent protein (BFP) that converts to green fluorescent protein (GFP) upon a C-to-T substitution as an assay to report directly on base editing activity within a cell. Using this assay, we optimize various base editing transfection parameters and delivery strategies. Moreover, we utilize this assay in conjunction with flow cytometry to develop a transient reporter for editing enrichment (TREE) to efficiently purify base-edited cell populations. Compared to conventional cell enrichment strategies that employ reporters of transfection (RoT), TREE significantly improved the editing efficiency at multiple independent loci, with efficiencies approaching 80%. We also employed the BFP-to-GFP conversion assay to optimize base editor vector design in human pluripotent stem cells (hPSCs), a cell type that is resistant to genome editing and in which modification via base editors has not been previously reported. At last, using these optimized vectors in the context of TREE allowed for the highly efficient editing of hPSCs. We envision TREE as a readily adoptable method to facilitate base editing applications in synthetic biology, disease modeling, and regenerative medicine.

Highlights

The rapid advancement of CRISPR/Cas-based technologies has allowed for the modification of human cells at precise genomic locations [1,2,3]
homology-directed repair (HDR) is inefficient in mammalian cells, especially in recalcitrant cells such as human pluripotent stem cells, and repair of double-stranded breaks (DSB) is predominantly achieved through non-homologous end joining (NHEJ) [5,6,7,8,9]
To establish that blue fluorescent protein (BFP) to green fluorescent protein (GFP) conversion could be used as the basis for an assay to detect genomic base editing, we utilized a BFP mutant that converts to a GFP upon a Cto-T nucleotide conversion (Figure 1A)

Summary

Introduction

The rapid advancement of CRISPR/Cas-based technologies has allowed for the modification (i.e. deletion, mutation and insertion) of human cells at precise genomic locations [1,2,3]. As an alternative to standard gene editing approaches that require a DSB, several groups have reported the development of deaminase base editors that do not rely on HDR to introduce single nucleotide genomic changes [10] Speaking, these base editors consist of a fusion of three components––a D10A nickase Cas endonuclease, cytidine deaminase (APOBEC1), and a DNA uracil glycosylase inhibitor (UGI). These base editors consist of a fusion of three components––a D10A nickase Cas endonuclease, cytidine deaminase (APOBEC1), and a DNA uracil glycosylase inhibitor (UGI) This complex is capable of converting cytosine to thymine [11] (or adenine to guanine on the complementary strand) [12] without the need for a DSB and homology repair template. Genome modification through the use of base editors has been shown to result in formation of fewer indels when compared to HDR-based methods [13,14]

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Conclusion