Massive Genome Editing Could Lead to Human Immunity to All Viruses

Base editing with reduced DNA nicking allows for the simultaneous editing of over 10,000 loci (13,000) in human cells.

Performing this feat in the human genome will require the modification of 4438 Amber codons. They have shown that gene editors that do not cause double- or single-strand DNA breaks can generate a number of edits sufficient to theoretically achieve this genome recoding and pave the way towards making pan-virus resistant human cells. This could have commercial application towards cell-based production of monoclonal antibodies, recombinant protein therapeutics, and synthetic meat production.

Researchers have overcome the toxicity limitation that prevented large-scale genome editing in human iPSCs and expanded the editing boundary by three orders of magnitude. The continued development of multiplex delivery along with non-toxic, high-efficiency DNA editors without DSBs or SSBs is paramount to the success of genome-wide recoding efforts to probe the inner workings of life, ultimately leading to the radical redesign of nature and ourselves.

More in-depth studies will be necessary, however, to assess the impact of this massive editing on normal cell processes, since collateral damage may occur. We expect the thorough on-and off-target analysis at repetitive elements to remain a difficult task to accomplish due to their high level of polymorphism; therefore, strong biological controls as well as new experimental and bioinformatics pipelines will be needed to overcome such a challenge.

Researchers developed a set of dead-Cas9 base editor (dBEs) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements – ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.

Improving the actual multiplexed eukaryotic genome editing capabilities by several orders of magnitude holds the potential of revolutionizing human healthcare. Combinatorial functional genomic assays would enable the study of complex genetic traits with applications in evolutionary biology, population genetics, and human disease pathology. Multiplex editing has also permitted the development of successfully engineered cell treatments, such as the chimeric antigen receptor (CAR) therapies, which require the simultaneous editing of three target genes. Future treatments may require many more modifications to augment cancer immunotherapies, slow down oncogenic growth, and reduce adverse effects, such as host-versus-graft disease. Furthermore, customizing host-versus-graft antigens in human- or nonhuman-donor tissues may require more modifications than have been made so far, for which the development of genome-wide editing technologies is needed. Special attention should be paid to the safety of the editing and its impact on the functional activity of the transplants, since donor tissues may persist in the patients for decades.

To complete genome-wide recoding and enable projects such as GP-write ultra-safe cells or the de-extinction efforts to regain lost biodiversity, safe DNA editors must be developed to increase the number of genetic modifications possible by several orders of magnitude without triggering overwhelming DNA damage, as well as overcoming the delivery of multiple distinct gRNAs per cell, the latter of which we do not address is this study.

Biorxiv – Enabling Large-scale Genome editing by reducing DNA Nicking