Universal toxin-based selection for precise genome engineering in human cells

Songyuan Li,Aleksandra Sieńska,Suman Mitra,Carl Möller,Xiufeng Xu,Sandra Wimberger,Silvia Cerboni,Matthew A Coelho,Giovanni Ciotta,Euan Gordon,Nina Akrap,Mohammad Bohlooly‐Y ,Benjamin Taylor ,Luna Simona Pane,Bojana Lazovic,Michelle J Porritt,Mike Firth,Anders Lundin,Giovanni Pellegrini,Marcella Sini,Grzegorz Sienski,Marcello Maresca

doi:10.1038/s41467-020-20810-z

Abstract

Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.

Highlights

Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools
The diphtheria toxin (DT) from Corynobacterium diphtheriae[30] is composed of domain B (DT-B) that binds to the membraneembedded form of heparin-binding EGF-like growth factor (HBEGF), and mediates endocytosis and translocation of DT
In this study, we leveraged the specific interaction between DT and its receptor HBEGF, and the toxin’s potency in inducing cell death[30] to develop a powerful co-selection genome editing system

Summary

Introduction

Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. While the accuracy of genome editing and the ability to predict off-target effects has been considerably improved, the efficiency of genetic engineering in somatic cells, especially precise substitutions and gene insertions, remains generally low, limiting potential therapeutic applications[7,8,9,10,11]. Cells that undergo simultaneous editing at the selection locus and a second-site targeted locus are typically enriched by fluorescent reporter-based sorting or resistance to specific cytotoxic reagents[14,15,16,17,18] This co-selection strategy has been used in cells from organisms ranging from Caenorhabditis elegans to humans[19,20,21,22,23]. The diphtheria toxin (DT) from Corynobacterium diphtheriae[30] is composed of domain B (DT-B) that binds to the membraneembedded form of heparin-binding EGF-like growth factor (HBEGF), and mediates endocytosis and translocation of DT

Methods

Results

Conclusion