Mutational scanning by multiplexed genome editing of the essential transcription termination factor Nrd1

Proteins operate through a few critical residues, yet most proteins remain uncharacterized at the deep molecular resolution, particularly within essential genes, where functional dissection is obstructed by lethality.

Here, we establish a platform for mutational scanning of essential genes at their endogenous locus , combining a repressible complementation system with multiplexed CRISPR-based genome editing in budding yeast. Our approach provides a generalizable framework for dissecting essential protein function in vivo , expanding the capacity to map critical residues underlying essential cellular processes. We applied this strategy to NRD1 , encoding an essential RNA Polymerase II (RNAPII) termination factor and performed a systematic alanine scanning with near-saturation coverage. We discovered novel and unexpected lethal mutations in the CTD-interacting domain (CID), thus revealing an unanticipated importance for this domain. Overall, our results demonstrate the power of our mutation scanning platform to map critical residues underlying essential cellular processes.