Over the past years, CRISPR/Cas genome editing has quickly become the method of choice for genetic manipulation, owing to its ease-of-use, flexibility, and efficiency. However, another main advantage of CRISPR technology is its scalability. In the study presented during this webinar, we used the high-throughput CRISPR-based Onyx platform to perform saturation mutagenesis on four different essential genes involved in cell envelope synthesis in E. coli. In total, 22,790 edits were designed so that every amino acid of these proteins would be replaced by every other amino acid. We used these saturation mutagenesis libraries to probe the essentiality of all different residues. Edits that could not be introduced point to mutations that do not support the viability and, hence, residues essential for protein function. This way, we were able to identify known essential amino acids, i.e. catalytic residues and residues involved in substrate binding, thereby validating our experimental approach. Additionally, studying the Onyx libraries' composition also pinpointed several residues that were previously not known to be essential. Besides offering vastly improved insights into protein function, we expect our results to help fight against antibiotic resistance. By providing insight into essentiality at an amino-acid level, we expect to aid structure-based drug design targeted against these essential proteins.

 

Learning Objectives