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Technology

This technology is a new, targeted method for easily generating massive mutant libraries. This technology focuses mutation on only the target protein-coding sequence, rather than increasing global hypermutation. Targeted mutation is performed using a fusion protein combining a processive DNA binding enzyme (T7 polymerase) with a mutagenic protein (APOBEC1). The resulting fusion protein moves along the target protein-coding DNA sequence and alters DNA along the length of the gene as it progresses. Importantly, the Apo-T7 fusion protein is targeted only to sequences with the proper flanking sequences, which eliminates off-target mutations. The low off-target mutational burden eliminates the toxicity normally associated with hypermutation and facilitates generation of much larger libraries than those made using global hypermutation. Additionally, elimination of off-target mutations reduces the number of false positives that need to be identified and discarded. In conclusion, this technology can generate directed evolution libraries of unprecedented size and quality that will lead to improved final products in a shorter amount of time.