UC Berkeley

A fundamental goal of protein biochemistry is to determine the sequence-function relationship, but the vastness of sequence space makes comprehensive evaluation of this landscape difficult. Advances in DNA synthesis and sequencing now allow researchers to assess the functional impact of thousands of amino acid substitutions in a single experiment, however, the quality and diversity of these mutations controls the breadth of knowledge gained by these emerging methods. Comprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. However, current techniques enabling comprehensive protein mutagenesis are based on PCR and require in vitro reactions involving specialized protocols and reagents. This has complicated efforts to rapidly and reliably produce desired comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space. Using the fluorescent protein iLOV as a model target, we build a complete mutagenesis library and find it to be specific and comprehensive, detecting 99.8% of our intended mutations. We then develop a thermostability screen and utilize our comprehensive mutation data to rapidly construct a targeted and multiplexed library that identifies significantly improved variants, thus demonstrating rapid protein engineering in a simple protocol.

Beyond simple amino acid substitutions, protein topology is also well-established as a key mechanism by which large, complex multi-domain proteins evolve highly specialized functions. While rationally constructed protein deletions have long been essential to elucidating biochemical properties, current techniques are insufficient for a comprehensive approach. Here we develop a method for constructing fitness landscapes for even the largest and most complex proteins, comprehensively surveying functional deletions in the RNA-guided DNA binding protein dCas9, the foundation for powerful genome editing and modifying technologies. CRISPR proteins are highly complex with numerous distinct domains responsible for activities such as guide RNA binding, DNA recognition, DNA unwinding, specificity sensing and ultimately the cleavage of each DNA strand. We exploit the fitness landscape to revert functionality and step backward in domain evolution, comprehensively minimizing dCas9 and screening for an essential function. We demonstrate the power of this technique by revealing the minimal RNA guided DNA binding module at 64% of the full CRISPR-Cas9 platform, providing many new opportunities for fusions and delivery. This exploration also uncovers evidence for a DNA unwinding mechanism in a domain heretofore viewed as dispensable in Cas9. These results highlight the power of comprehensive protein deletions to clearly elucidate the boundaries of a central function.

Together, amino acid substitution and topological mutation (encompassing deletions, insertions, and circular permutations) comprise all possible genetic protein modifications. This work has served to develop simple and robust methods, which remain programmable and comprehensive, for both substitution and topological mutagenesis. The construction of high-quality protein libraries is a foundational step for applications in the fundamentals of protein biochemistry, disease prediction, and protein engineering. Ultimately, understanding the general principles of protein sequence-function landscapes - enabled by massively parallel experimentation - will allow computational methods to synergize with programmable mutagenesis and vastly improve the search for novel fitness variants.