This technology is a directed evolution hypermutation technique that has applications in biopharmaceutical development and life science research.
Biomolecules, such as proteins, have diverse molecular functions that can be engineered for research and therapeutic uses. However, it remains challenging to rationally design protein functions due to the complex three-dimensional structure of proteins. Inspired by nature, scientists have developed directed evolution techniques that can quickly modify proteins to have desired specificity, stability, or binding affinity characteristics. Directed evolution requires generation and screening of large libraries of mutant proteins for desired characteristics. Current hypermutation techniques for generating mutant libraries in vivo are labor intensive, inefficient, and result in widespread, untargeted mutation. Additionally, these techniques generate many off target mutations outside the target sequence, which results in cellular toxicity and frequent false positives. There is therefore a need to develop user-friendly and efficient techniques for generating mutant libraries.
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.
- Directed hypermutation reduces off-target effects and toxicity
- Increased mutant library size
- Inducible expression for customized mutagenesis duration
- Better final protein products in a shorter amount of time
- Fewer false positives