Deep mutational scanning of the RNase III-like domain in the Trypanosoma brucei RNA editing protein KREPB4

Kinetoplastid pathogens including Trypanosoma brucei, T. cruzi, and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degron-controlled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. As proof-of-principle, we used the method to query the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of our method over traditional sequence homology searching to identify functional residues.