An evolved, orthogonal ssDNA generator for targeted hypermutation of multiple genomic loci.

Achieving targeted hypermutation of specific genomic sequences without affecting other regions remains a key challenge in continuous evolution. To address this, we evolved a T7 RNA polymerase (RNAP) mutant that synthesizes single-stranded DNA (ssDNA) instead of RNA in vivo, while still exclusively recognizing the T7 promoter. By increasing the error rate of the T7 RNAP mutant, it generates mutated ssDNA that recombines with homologous sequences in the genome, leading to targeted genomic hypermutation.

Enhancing Cellular and Enzymatic Properties Through In Vivo Continuous Evolution.

Directed evolution seeks to evolve target genes at a rate far exceeding the natural mutation rate, thereby endowing cellular and enzymatic properties with desired traits. In vivo continuous directed evolution achieves these purposes by generating libraries within living cells, enabling a continuous cycle of mutant generation and selection, enhancing the exploration of gene variants. Continuous evolution has become powerful tools for unraveling evolution mechanism and improving cellular and enzymatic properties.

Integrating Deep Learning and Synthetic Biology: A Co-Design Approach for Enhancing Gene Expression via N-Terminal Coding Sequences.

N-terminal coding sequence (NCS) influences gene expression by impacting the translation initiation rate. The NCS optimization problem is to find an NCS that maximizes gene expression. The problem is important in genetic engineering.