Sequence to Activity Mapping: Computational Approaches and Experimental Tools

We developed a method for rapidly investigating sequence-to-activity relationships in a large scale, at the genomic level of E. coli. This method combines phage-based recombineering with CRISPR genome editing: site-specific gRNAs and editing templates are synthesized in parallel on a microarray and later pool-cloned into a dedicated plasmid. The gRNA, together with an inducible Cas9 digest the genome in a sequence-specific manner, while the editing template is integrated genomically by the recombineering machinery. The editing template harbors two mutations: the first is the desired modification and the second silently mutates a neighboring PAM sequence, which is necessary for CAS9 cleavage. This approach allows to build genome-wide libraries and complete saturation of whole genes. Since the plasmid editing templates highly correlates with the genomic edits, simple plasmid sequencing replaces whole genome interrogation, making this system easily trackable. We demonstrate CREATE by completely saturating the essential E. coli folA gene, generating its full sequence to activity map. Moreover, we also challenged these library cells with trimethoprim, a FolA specific inhibitor, and found two mutations within the same site that confer resistance to this drug.

We previously reported on a new approach for saturation mutagenesis. In contrast to the commonly used NNK codon, we compute the minimal set of degenerate codons to exactly cover the desired set of amino acids. Importantly, the compressed codons do not include stop codons, and cover every amino acid only once, eliminating redundancy. Moreover, the codons selected are with high usage scores, as defined by the target organism. We now report a dedicated website that includes all these and more functionalities, improving accessibility and ease of use for the synthetic biology and bioengineer’s community.