Aug 2026· Biomedicines· Vol 14· 0 citations· 95 references
Medicine
TL;DR
In this review, a review of recent in vivo hypermutation tools that enable rapid sampling of the vast evolutionary landscape, all while supporting simultaneous selection of the best proteins within living organisms are discussed.
Abstract
Directed evolution has revolutionized protein engineering by applying the principles of natural selection to the laboratory. However, traditional in vitro methods are quite labor-intensive, while common in vivo methods suffer from low mutation rates and high rates of off-target mutations. To address these issues, researchers have developed targeted mutagenesis tools for rapid in vivo evolution of biomolecules. In this review, we discuss recent in vivo hypermutation tools that enable rapid sampling of the vast evolutionary landscape, all while supporting simultaneous selection of the best proteins within living organisms. We focus on three main mechanisms of hypermutation: (i) orthogonal replication, which uses error-prone replication machinery to replicate the target gene with low fidelity; (ii) CRISPR-Cas-guided mutators, where mutagenic proteins are localized to virtually any user-defined loci; and (iii) transcription-coupled mutagenesis, a simple, yet elegant tool that exploits the innate processivity of orthogonal ribonucleic acid (RNA) polymerases to guide mutagenic proteins along the target gene during transcription. We highlight key advantages of these systems, as well as some clinically- and biotechnology-relevant applications. We discuss important limitations and how they could be addressed in the future to make hypermutation tools with broad mutational spectra and windows that span entire genes with minimal off-target effects.
A PLM-assisted physics-driven approach is presented that utilizes atomistic molecular dynamics simulations and automated path searching to efficiently obtain the complete kinetic insights, including the transition state structures, for the conformational changes of Cas before DNA cleavage.
Sequence-programmable directed evolution systems have great potential to accelerate bioengineering. Diversity-generating retroelements (DGRs) are natural hypermutation systems widely distributed in prokaryotes and bacteriophages with the capacity to introduce diverse mutations at template-specified sites of target genes. Here, we show that DGRs can be installed in E. coli and reprogrammed for the continuous, iterative mutagenesis of user-defined target genes. We show that the DGR template RNA can be reprogrammed for gene- and residue-specific mutagenesis, leaving untargeted, adjacent residues unchanged. Furthermore, we establish continuous DGR-enabled mutagenesis with conjugation-mediated horizontal gene transfer of target genes (HGT-DGR) into a new host for the progressive accumulation of target-specific mutations. Iterative HGT-DGR mutagenesis over seven cycles yielded an average mutation load of approximately 6% across adenine positions in the target segment, generating a diverse library of variants comprising 40% mutant sequences, with a median pairwise Hamming distance of 4 among mutant variants. HGT-DGR enables iterative diversification of either the same or different user-specified segments of the target gene, as demonstrated with the directed evolution of the M. mazei pyrrolysyl-tRNA synthetase for non-canonical amino acid incorporation. HGT-DGR provides a simple, low-cost, sequence-programmable system that enables iterative, position-specific and tunable in vivo mutagenesis of any target sequence for applications in biotechnology and medicine.
Yang Liu, Yang-Qi Gu, Ganesh Agam et al.· bioRxiv· 0 citations
The rapid advancement of protein engineering and genetic code expansion technologies over the last decade has reshaped how researchers rationally design proteins with novel catalytic functions. Among these approaches, the site-specific incorporation of unnatural amino acids has enabled the introduction of chemical functionalities that are inaccessible to the canonical amino acid space. In this perspective, we highlight the metal-chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) and its emerging utility in mediating nucleic acid cleavage. Multiple studies have demonstrated the successful site-specific incorporation of BpyAla into proteins of interest, where subsequent metal coordination enables catalytic cleavage of DNA and RNA substrates. Here, we discuss the potential of BpyAla-mediated nucleic acid cleavage, with emphasis on the development of next-generation BpyAla analogues, the exploration of alternative metal cofactors, cooperative and multi-residue design strategies, and the expansion of compatible protein scaffolds and nucleic acid substrates. Designable BpyAla-engineered systems represent an emerging frontier in artificial metallonuclease design, with potential long-term relevance to targeted nucleic acid therapeutics.
E. Lundrigan, Matthew T. O’Neill, J. P. Pezacki· Frontiers in Chemical Biolog...· 0 citations
The programmed diversification of genes and other encoded genetic elements, through site directed- and site saturation-mutagenesis, underpins approaches to learning the relationship between DNA sequence and function, and forms a foundation for creating new function through directed evolution. However, current approaches for generating large genetic libraries commonly generate diversity in vitro and then transform the resulting library into cells; this multistep process is inefficient and this paradigm places limits on the scale of diversity that can be achieved and the size of diversified genetic elements that can be introduced into cells. Here we demonstrate a distinct paradigm for library generation through: 1) efficient transfer of genetic elements, as cargos in F plasmids, to recipient cells bearing libraries of retron editors, 2) efficient editing of cargo genes in recipient cells, and 3) continuous iteration of the conjugation-editing cycles with selection for recipients in sequential cycles using three selection markers in series. In this paradigm, the library diversity emerges multiplicatively through the iteration of conjugation-editing cycles. Using this paradigm, we generated substantial libraries that enabled the selection of new phenotypes, with library members containing up to six distinct edits and edits arising from several conjugation-editing cycles.
Fabian B. H. Rehm, Martin Spinck, Jason W. Chin· bioRxiv· 0 citations
Genomic manipulation has advanced from stochastic nuclease‐mediated disruption toward programmable, deterministic precision. Early clustered regularly interspaced short palindromic repeats (CRISPR) strategies enabled targeted mutagenesis through double‐strand breaks; however, their therapeutic application is limited by genotoxicity, chromosomal instability, and dependence on endogenous repair pathways that are difficult to predict. In this review, we examined the transition from gene editing to genome writing, an approach that decouples genomic modification from host repair pathways to better balance efficiency, precision, and payload delivery. We also discussed the principles of precision technologies, including base and prime editors, and described emerging large‐scale writers, such as CRISPR‐associated transposases and recombinase‐based bridge RNAs, which enable the integration of multi‐kilobase synthetic modules. Beyond enzymatic mechanisms, we further considered the combined use of generative artificial intelligence, structural biology, and novel delivery architectures as potential strategies to overcome current biological limitations. Taken together, these developments point toward Generative Biology, in which computational design and high‐throughput screening transform the genome from a static substrate into a more dynamic model for complex, synthetic functional design.