Universal toxin-based selection for precise genome engineering in human cells.
Songyuan LiNina AkrapSilvia CerboniMichelle J PorrittSandra WimbergerAnders LundinCarl MöllerMike FirthEuan GordonBojana LazovicAleksandra SieńskaLuna Simona PaneMatthew A CoelhoGiovanni CiottaGiovanni PellegriniMarcella SiniXiufeng XuSuman MitraMohammad Bohlooly-YBenjamin J M TaylorGrzegorz SienskiMarcello MarescaPublished in: Nature communications (2021)
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.
Keyphrases
- crispr cas
- genome editing
- induced apoptosis
- induced pluripotent stem cells
- cell cycle arrest
- endothelial cells
- escherichia coli
- single cell
- endoplasmic reticulum stress
- cell therapy
- gene expression
- oxidative stress
- signaling pathway
- stem cells
- cell proliferation
- type diabetes
- metabolic syndrome
- cell free
- high throughput
- single molecule
- skeletal muscle
- papillary thyroid
- young adults
- pluripotent stem cells
- pi k akt
- adipose tissue
- bone marrow
- solid state
- genome wide identification
- mesenchymal stem cells