Virus-free continuous directed evolution in human cells using somatic hypermutation

Random mutations followed by natural selection is one of the key mechanisms during natural evolution that results in the generation of new functions. This process is typically slow in nature because of low mutational frequency. In contrast, the B cells of mammalian immune systems have evolved somatic hypermutation (SHM) mechanisms that introduce mutations at the immunoglobulin genomic loci at a significantly higher frequency than the rest of the genome. SHM allows B cells to rapidly evolve new antibody sequences without compromising their fitness as a consequence of genome-wide mutations. In this work, we developed a continuous directed evolution platform in human B cell lines (CODE-HB) that recruits and repurposes the SHM mechanisms to rapidly evolve reporter proteins like the Green Fluorescent Protein. This approach uses a stable, non-immunoglobulin locus within the genomes of human B cell lines. To comprehensively characterize the mutational profile and breadth of this strategy, we performed single-molecule sequencing experiments. We illustrate the utility of the platform by rapidly evolving antibody fragments in a continuous manner by B cell surface display targeting avian subtypes of influenza hemagglutinin (e.g., H5) from emerging strains.