Creating multiple-crossover DNA libraries independent of sequence identity

We have developed, experimentally implemented and modeled in silico a methodology named SCRATCHY that enables the combinatorial engineering of target proteins, independent of sequence identity. The approach combines two methods for recombining genes: incremental truncation for the creation of hybrid enzymes and DNA shuffling. First, incremental truncation for the creation of hybrid enzymes is used to create a comprehensive set of fusions between fragments of genes in a DNA homology-independent fashion. This artificial family is then subjected to a DNA-shuffling step to augment the number of crossovers. SCRATCHY libraries were created from the glycinamide-ribonucleotide formyltransferase (GART) genes from Escherichia coli (purN) and human (hGART). The developed modeling framework eSCRATCHY provides insight into the effect of sequence identity and fragmentation length on crossover statistics and draws contrast with DNA shuffling. Sequence analysis of the naive shuffled library identified members with up to three crossovers, and modeling predictions are in good agreement with the experimental findings. Subsequent in vivo selection in an auxotrophic E. coli host yielded functional hybrid enzymes containing multiple crossovers.